The authors gratefully acknowledge their colleagues Jeffrey Tom and Aimin Song for peptide synthesis, Nianfeng Ge for TMA construction, Shari Lau for IHC staining, Melissa Edick for digital microscopic scanning, and Hai Ngu for digital image quantification.

1. Preparation of stock solution and tools
<ol>
	<li>Prepare 20 mL of a 25% w/v BSA solution by mixing 5 g BSA powder in 14 mL of PBS, pH 7.2 in a 50 mL conical tube until evenly dispersed. Vortex as necessary to disperse the BSA powder.
	<ol>
		<li>Keep the solution overnight at 4 &#176;C to allow complete dissolution. Adjust the final volume to 20 mL with PBS to make a 25% w/v stock solution.</li>
	</ol>
	</li>
	<li>Prepare 20 mL of a 31.3% w/v BSA solution by mixing 6.26 g BSA powder in 13 mL of PBS, pH 7.2 in a 50 mL conical tube until evenly dispersed. Keep the solution overnight at 4 &#176;C to allow complete dissolution. Adjust the final volume to 20 mL with PBS to make a 31.3% w/v stock solution.</li>
	<li>Preheat a heat block to 85 &#176;C. 
	NOTE: The protocol below creates peptide/BSA gels with volumes of 1.26-1.4 mL formed in 1.5 mL microcentrifuge tubes. To use smaller volumes, for example, when antigen stocks are limiting, prepare the gels in PCR tubes and use a thermocycler set to 85 &#176;C as a heat block.</li>
	<li>Test that the BSA/formaldehyde mixture forms a gel as expected by mixing 700 &#181;L of 25% BSA solution with 700 &#181;L of 37% formaldehyde. Mix well by pipetting up and down 5 times within 5-10 s. Avoid creating air bubbles. 
	CAUTION: Concentrated formaldehyde is toxic; use with appropriate safety precautions.</li>
	<li>Immediately after mixing the BSA and formaldehyde solutions, place the closed microcentrifuge tube in a heat block at 85 &#176;C for 10 min. Remove the tube from the heat block and allow it to cool. Confirm that the gel has formed as expected.</li>
</ol>
2. Preparation and dilution of peptides
<ol>
	<li>Obtain 5-20 mg of lyophilized peptide of the desired sequence. 
	NOTE: The C-terminal 16 amino acids of the human ERBB2 intracellular domain recognized by 4B5 is TPTAENPEYLGLDVPV-COOH.
	<ol>
		<li>Add 4 amino acids to the N-terminus, acetyl-YGSG, and C-terminus, GSGC-amide to facilitate cross-linking of the peptide to BSA and provide spacing between the BSA molecule and the peptide epitope. 
		NOTE: The complete sequence is: acetyl-YGSGTPTAENPEYLGLDVPVGSGC-amide.</li>
		<li>If desired, use other N- and C-terminal amino acid sequences to extend the core peptide epitope. 
		NOTE: The impact of different sequences varies with different antibody/epitope combinations. The addition of the C-terminal peptide reduces the binding of some antibodies to C-terminal epitopes. In such cases, omit this sequence.</li>
		<li>Confirm that peptides from commercial sources are supplied at &#62;95% purity, the composition of which is confirmed by HPLC and mass spectrometry analysis.</li>
	</ol>
	</li>
	<li>Calculate the necessary volumes for the 5x (1.25 E-2 M) peptide stock solutions. Referring to Table 1, columns C-E, enter values for the antigen molecular weight (g/mole), percent antigen purity (0-100), and antigen mass (mg). 
	NOTE: The volume of solvent (in &#181;L) to resuspend the sample to achieve a stock solution of 1.25 E-2 M is 800 x antigen molecular weight x percent antigen purity/antigen mass.
	<ol>
		<li>Prepare and clearly label eight 1.5 mL microcentrifuge tubes. 
		NOTE: The tubes will contain 5x peptide stock in a solvent, 1x peptide stock in a solvent, five 10x serial dilutions of 2.5 E-4 M to 2.5 E-8 M peptide/BSA/formaldehyde gel, and a negative control gel containing BSA/formaldehyde lacking added antigen. All gel samples look identical. When preparing multiple sets of peptide dilutions at one time, take care to label and identify all tubes and processing cassettes correctly. Use color-coded microcentrifuge tubes and processing cassettes where possible to minimize misidentification.</li>
	</ol>
	</li>
	<li>Prepare a 5x peptide stock solution at 1.25 E-2 M by resuspending the entire mass of lyophilized peptide (20 mg for the ERBB2 peptides) in 60 &#181;L of the appropriate solvent. 
	NOTE: In this example, dimethylformamide (DMF) was added directly to the vendor&#39;s container.
	<ol>
		<li>Inspect the solution to ensure that the peptide is completely dissolved. If necessary, add additional solvent and/or sonicate the sample until the peptide is completely dissolved, taking care not to exceed the volume calculated in Table 1 for the 5x peptide stock. 
		CAUTION: DMF is toxic; use with appropriate precaution. 
		NOTE: Depending on the amino acid sequence, and the corresponding hydrophobicity and charge, peptides may be soluble in DMF, dimethyl sulfoxide (DMSO), pure water, or dilute solutions of acetic acid, formic acid, or ammonium bicarbonate. Peptide characteristics may be calculated using a variety of online tools17. Some peptide vendors may suggest solvents appropriate for specific sequences.</li>
		<li>Add solvent as necessary to bring the volume of the 5x peptide stock to the final volume calculated in Table 1. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s. Peptide stock solutions can be stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Referring to Table 2, Column C, prepare 150 &#181;L of 1x peptide stock solution (2.5 E-3 M) by diluting 30 &#181;L of the 5x peptide stock into 120 &#181;L of solvent (DMF this example). Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s.</li>
	<li>Referring to Table 2, Column D, prepare 700 &#181;L of 5 E-4 M peptide/BSA solution (Dilution 1) by diluting 140 &#181;L of 1x peptide stock into 560 &#181;L of 31.3% BSA/PBS, pH 7.2. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s. 
	NOTE: The final BSA concentration of this solution is 25% (w/v).</li>
	<li>Referring to Table 2, Columns E-H, prepare four successive 10x serial dilutions of the 5 E-4 M peptide/BSA stock by adding 70 &#181;L of peptide/BSA solution to 630 &#181;L of 25% BSA/PBS, pH 7.2. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s. 
	NOTE: After this step, there will be five 10-fold serial dilutions of peptide (5 E-4 M to 5 E-8 M) in 25% BSA/PBS, pH 7.2. The first four samples will contain 630 &#181;L. The last sample will contain 700 &#181;L.</li>
	<li>Referring to Table 2, Column I, prepare a negative control BSA sample containing 700 &#181;L of 25% BSA/PBS, pH 7.2 (Figure 1A).</li>
</ol>
3. Preparing BSA-peptide gels
<ol>
	<li>Confirm that the heat block or thermocycler is stable at 85 &#176;C.</li>
	<li>Refer to Table 3, Columns B-E. Working one sample at a time, add to the first 25% BSA/peptide sample (Dilution 1) 630 &#181;L of 37% formaldehyde. Mix well by pipetting up and down 5 times within 5-10 s. Avoid creating air bubbles. 
	CAUTION: Concentrated formaldehyde is toxic; use with appropriate safety precautions.
	<ol>
		<li>After mixing the peptide/BSA and formaldehyde solutions, place the closed microcentrifuge tube in a heat block at 85 &#176;C for 10 min. 
		NOTE: Mix the BSA-peptide solution and formaldehyde thoroughly, but do not spend more than 10 s pipetting the mixture before placing the sample on heat. Since formaldehyde cross-linking begins immediately, the gel may form differently if the procedure is varied for different samples. The final BSA concentration in these gels is 12.5% (w/v). Final BSA concentrations less than 10% may yield gels that do not solidify; final BSA concentrations greater than 16% may produce gels more brittle and difficult to section after processing.</li>
		<li>Repeat steps 3.2 and 3.2.1 for each of the dilutions 2-4.</li>
		<li>Repeat steps 3.2 and 3.2.1 for dilution 5, but add 700 &#181;L of 37% formaldehyde, a volume equal to the 700 &#181;L of BSA-antigen solution.</li>
		<li>Refer to Table 3 column Column G; repeat steps 3.2 and 3.2.1 for the negative control sample, adding 700 &#181;L of 37% formaldehyde, a volume equal to the 700 &#181;L of negative control BSA solution.</li>
	</ol>
	</li>
	<li>Remove the tubes from the heat block after 10-12 min. The heating time should be as consistent as possible for each sample. Allow the gels to cool on the benchtop for 5-10 min (Figure 1B).</li>
	<li>Using a clean, flexible disposable laboratory spatula, remove the gel sample in one piece from the microcentrifuge tube, and place it in a sealed container containing at least 15 mL of neutral buffered formalin (NBF), using a separate container of NBF for each sample.
	<ol>
		<li>Alternatively, cut off the bottom of the microcentrifuge tube with a new single edge razor blade, and push the gel out from the bottom with air or a suitable probe (Figure 1C-G). 
		&#8203;NOTE: The solidified formaldehyde/BSA gels can remain in the microcentrifuge tubes at room temperature for up to 24 h. Leaving the gels in the microcentrifuge tube for more than 24 h can cause them to become brittle and more difficult to process and section.</li>
	</ol>
	</li>
</ol>
4. Trimming, processing, and embedding BSA gels
<ol>
	<li>Trim the gel cone into cylindrical discs approximately 5 mm thick using a clean single edge razor (Figure 1H,I). Wrap the discs in a biopsy wrap, placing one larger gel disc into one cassette (to be used in the pilot study in step 5), and the remaining gel discs together into a second cassette (Figure 2A,E) for use in tissue microarray (TMA) construction in step 6. Place the wrapped gel discs in clearly labeled tissue processing cassettes.
	<ol>
		<li>Place the cassetted gels in at least 15 mL of 10% NBF per gel sample before processing, using a separate container of NBF for each sample. Gels can remain in 10% NBF for 6-48 h.</li>
	</ol>
	</li>
	<li>Process the gels in an automated histology tissue processor, following a large tissue schedule with pressure and vacuum. Each step takes 1 h: 10% NBF, 70% ethanol, 95% ethanol (repeat two times), 100% ethanol (repeat two times), xylenes (repeat three times), paraffin at 60 &#176;C (repeat three times). 
	NOTE: For investigators choosing to process samples manually, follow the same sequence of reagents and times.</li>
	<li>When the sample processing is completed, remove the cassettes from the tissue processor and move them to the paraffin embedding center.</li>
	<li>Unwrap gels from the biopsy wrap and embed the gels in paraffin. For each sample, embed one disk of gel in a small 15 mm x 15 mm mold (Figure 2B-D), and the remaining gel discs together in a second larger mold (Figure 2F-H). The first block with one sample will be used to test the peptide gel in a pilot study. The second block can be used for TMA construction.</li>
</ol>
5. Pilot evaluation of the peptide dilution series
<ol>
	<li>For each peptide dilution series, plan to create two glass slides containing a total of 6 separate sections: one section from each of the five dilution series samples, plus one section from the BSA-only negative control sample.
	<ol>
		<li>Onto the first glass slide, cut one 4 &#181;m thick section from each of the smaller blocks containing one gel disc with the three highest peptide concentrations (2.5 E-4 M to 2.5 E-6 M).</li>
		<li>Onto a second slide, cut one 4 &#181;m thick section from each block with the two lowest peptide concentrations (2.5 E-7 M and 2.5 E-8 M) and one section from the BSA-only control block. Record the order of the samples on the slides. 
		NOTE: Expect the paraffin-embedded gels to cut smoothly, producing uniform sections without fragmentation, tearing, or chattering artifact. If particular paraffin-embedded gel samples are difficult to section, briefly soak the block face in ice-cold distilled water before sectioning. If necessary, experiment with different soaking times or with different solutions (e.g., ammonia water).</li>
		<li>After sectioning, dry the slides at room temperature (about 23 &#176;C) for 24 h followed by 60 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Stain the two slides prepared for each peptide with the desired antibody according to standard IHC protocols. 
	NOTE: The primary antibody on-slide concentration used for rabbit monoclonal 4B5 in this demonstration was 1.5 ug/mL.
	<ol>
		<li>Expect to see a relatively uniform signal within each gel section, with the different gel samples showing a range of signal intensity corresponding to the peptide dilutions.</li>
	</ol>
	</li>
	<li>If the results for the pilot study are satisfactory, construct a TMA from the gel donor blocks containing different concentrations of peptide antigen, as described in the next steps of the protocol.</li>
</ol>
6. BSA gel TMA construction
<ol>
	<li>Construct a tissue microarray containing duplicate 1 mm diameter cores from donor blocks containing BSA gel alone and BSA gels containing all five dilutions of ERBB2 peptide. 
	NOTE: If desired, include BSA gels containing the same five dilutions of a non-target peptide as additional negative controls. If desired, include cores of representative ERBB2-expressing cell lines as positive controls.</li>
	<li>Cut 4 &#181;m thick sections of the TMA and stain with 1.5 ug/mL of anti-ERBB2/HER2/neu rabbit monoclonal 4B5 (see Table of Materials) according to laboratory-standard protocols.</li>
	<li>Assess the resulting stain intensity qualitatively by inspection or quantitatively by digital image scanning and analysis (Figure 3A,B). 
	NOTE: As digital image analysis is not the focus of this protocol, these steps are left for the reader to perform according to their preference.</li>
</ol>

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Charles A. Havnar, Kathy J. H&#246;tzel, Charles A. Jones, Carmina M. Espiritu, Linda K. Rangell, and Franklin V. Peale are employees and stockholders of Genentech and Roche. Their affiliates produce reagents and instruments used in this study.

This method allows the user to create uniform samples of known composition and antigen concentration as standards in IHC reactions, using materials and techniques familiar to most histology laboratories. The most crucial step is to identify the epitope to which the antibody of interest binds. This protocol describes using a linear peptide antigen from the ERBB2/HER2 ICD. The same protocol can be used to form BSA gels containing oligonucleotides, fluorescent labels, protein domains, or full-length proteins. This latter approach can be helpful for antibodies binding to conformational epitopes that are not recreated by a single linear peptide sequence. For example, BSA gel standards containing 0.1 mg/mL naive IgG from mice, rats, and rabbits may be used as process controls to confirm that secondary detection and staining steps have worked as expected.
The antigen-BSA gel method described here complements other techniques to control and standardize IHC reactions. Cell lines and tissue samples with well-characterized expression levels of target antigens have been essential to well-controlled IHC protocols1,3,4. Peptides coupled to the surface of glass slides and glass beads have been proposed as quantitative controls7,8,9. Each of the potential methods has overlapping advantages and limitations. The BSA-antigen gels have several advantages.&#160;They are simple to make and adaptable to various antigen compositions and concentrations. They reproduce some of the three-dimensional architecture of tissue samples while controlling for the inherent heterogeneity found in biological samples. Because the synthetic antigen gels can be made with user-selectable antigen concentrations extending from below the limit of detection to the highest concentrations found in biological samples (~10-4 M)2, they offer opportunities to calibrate and standardize assays performed with modern techniques, e.g., AQUA18 and imaging mass spectrometry19, whose dynamic range far exceeds traditional chromogenic IHC. In addition, the method allows controls incorporating more than one antigen, which can be used in multiplex assay development and standardization.
While the method requires the user to know the epitopes for the antibodies of interest, many antibodies bind to well-documented linear epitopes. Epitope mapping techniques20,21,22 can often identify undefined linear epitopes, and synthetic peptides&#160;including known linear epitopes can be purchased at a modest cost. Other antibodies bind to conformational epitopes that are not reproduced by linear peptides but are preserved in whole proteins or protein domains. For some of these antibodies, recombinant forms of the target antigens are commercially available.
For creating control samples with optimal uniformity and reproducibility, the peptide or other target antigen must be completely dissolved and carefully diluted into BSA solutions. It is also essential that efficient, rapid mixing of the BSA/peptide solution with 37% formaldehyde occurs before the sample starts to gel, and that the mixed sample is then placed promptly at 85 &#176;C for 10 min. For avoiding over-fixation and processing artifacts, gels should be taken through processing and paraffin embedding according to the recommended schedule.
Variations on the method described here may be helpful in specific contexts. For instance, alternative N-and C-terminal peptide sequences may be used to optimize the detection of peptides bound to BSA2. It should be anticipated that antibodies recognizing epitopes from the extreme C-termini of proteins may be variably sensitive to C-terminal modifications of the native sequence. The peptide-BSA gel samples may also be formed by heating at 85 &#176;C for 10 min without fixative, and then prepared as frozen blocks or post-fixed with a variety of chemistries2. In addition, peptide conjugation can be accomplished with maleimide chemistry or other methods than described here.
Biological samples such as cell lines and tissues have the advantage as controls of representing the complexity of the many variables found only in life. On the other hand, because tissue and cell standards are both internally heterogeneous and variable from sample to sample, the interpretation of variable staining from run to run is confounded. In addition, the antigen concentrations in tissues and cells are often known only qualitatively, though, with the increasing use of quantitative mass spectrometry, absolute protein concentrations are reported more frequently23. The BSA-antigen controls described here intentionally eliminate the spatial and biological context of proteins in cells and tissues. For this reason, the correlation between IHC signal intensity and antigen concentration found in these controls may imperfectly reproduce that seen in tissues. The synthetic controls described here may be helpful to optimize and standardize some aspects of IHC assay performance. Still, they do not prevent the need for the appropriate use of other control samples. For instance, potential non-specific staining in a target tissue can be evaluated only by the parallel use of a negative tissue control sample. In addition, BSA-antigen gel controls do not reproduce tissue-specific pre-analytic variables, including warm and cold ischemia time, proteolysis, or fixation and processing conditions that may affect IHC performance. Accordingly, and as has been discussed elsewhere in more detail1,3,4, investigators should make thoughtful use of cell and tissue standards to precisely characterize the performance of the IHC system.
BSA-antigen controls may be used to develop or optimize staining protocols and serve as intra- and inter-laboratory reference samples when assessing the reproducibility of established protocols. Access to well-defined standard samples should allow users to more rigorously characterize IHC reaction behavior, identify variation in staining performance, and optimize reaction conditions to achieve specific objectives.

Immunohistochemistry (IHC) allows the sensitive and specific, spatially precise detection of target antigens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, IHC staining results may be affected by multiple variables, including warm and cold ischemia time, tissue fixation, tissue pretreatment, antibody reactivity and concentration, assay detection chemistry, and reaction times1. Accordingly, reproducible performance and interpretation of IHC reactions require rigorous control of these variables and the use of well-characterized positive and negative control samples. Frequently used controls include paraffin-embedded tissues or cultured cell lines known from independent analyses to express the antigen of interest, but such samples are not always available1. Furthermore, the expression levels of the target antigens in tissues and cell line controls are generally understood only qualitatively and may be variable. Controls containing reproducible, precisely known concentrations of target antigen can assist in the optimization of IHC reaction conditions. A general method, adaptable to a variety of antigen types in a physiologically relevant range of concentrations to create synthetic IHC control samples, has been described by the authors2. A detailed protocol is provided here for the creation and use of this type of standard.
Appropriate controls are essential for the valid interpretation of IHC assays1,3,4. Tissues, cultured cells, and peptide-coated substrates have been employed as IHC controls according to the investigators&#39; specific needs. The advantages and limitations inherent in using tissues as IHC controls have been extensively discussed1,4. For many antibodies, appropriate controls can be chosen from selected normal tissues containing cell populations expressing the target antigen over a wide dynamic range. Tissue controls are less suitable when the target antigen is not well-characterized concerning expression site or abundance, or when potentially cross-reacting antigens are co-expressed in the same cells or tissue sites. In these contexts, blocks of cultured cell lines expressing the antigen of interest can be helpful. For providing further evidence of target specificity, cell lines can be engineered to over-or under-express target antigens. For example, such an approach was recently used to evaluate a variety of anti-PD-L1 assays using a tissue microarray of isogenic cell lines expressing a range of PD-L1 antigen5. Practical limitations to the routine use of cell line blocks include the cost and time needed to produce sufficient cell numbers and the fact that the expression of some antigens may not be reliably consistent, even within clonal cell lines6. Synthetic peptides are a third option for IHC controls for antibodies that recognize short linear epitopes. Steven Bogen and colleagues have published extensively on the use of peptides coupled to the surface of glass slides7,8 and glass beads9. One study by this group demonstrated that quantitative analysis of peptide-based IHC controls could detect staining process variation missed by qualitative evaluation of tissue controls analyzed in parallel10. While standards using bead-based antigens could be widely applicable, many details are proprietary to the authors, limiting widespread adoption.
Another approach to IHC standards incorporates target antigens into artificially created protein gels. This concept was first demonstrated by Per Brandtzaeg in 1972 in a study in which normal rabbit serum was polymerized using glutaraldehyde11. Small blocks of the resulting gel were then soaked for 1-4 weeks in solutions containing the immunoglobulin antigens of interest at various concentrations. After alcohol fixation and paraffin embedding, sections of the resulting controls were shown to stain with intensities corresponding to the logarithm of the antigen solutions in which they had been soaked. Later, investigators prepared glutaraldehyde conjugates of specific amino acids in dilute BSA or brain homogenate solutions as positive controls in immune-electron microscopy studies12,13. More recent work investigated the use of gels made from formaldehyde-fixed protein solutions as surrogates for FFPE tissue in mass spectrometry analysis14. Another recent work investigated the structure and properties of gels formed by heating human or bovine serum albumin solutions at various concentrations and pH15. These authors found that serum albumin forms three types of gels differing in mechanical elasticity, secondary structure preservation, and fatty acid-binding capability depending on the experimental conditions. Together, these papers demonstrate the general feasibility of the approach employed here. Protein solutions of defined composition create tissue-like gels that can be further processed, sectioned, and stained using routine histological methods.
This protocol describes the formation of a synthetic IHC control made from bovine serum albumin (BSA) polymerized with heat and formaldehyde. The gels can incorporate a wide variety of antigens, including full-length proteins, protein domains, and linear peptides, as well as non-protein antigens&#160;including oligonucleotides2. This demonstration uses an example antigen&#160;a linear peptide encoding the C-terminal 16 amino acids of the human ERBB2 (HER2/neu) protein TPTAENPEYLGLDVPV-COOH (see Table of Materials). This sequence includes the epitopes recognized by three commercially available, diagnostically relevant antibodies&#160;including the Herceptest polyclonal reagent (ENPEYLGLDVP) and the monoclonal antibodies CB11 (AENPEYL) and 4B5 (TAENPEYLGL) (see Table of Materials)16.
The method demonstrated here employs readily available reagents using processes and techniques familiar to any practicing histology laboratory. The most significant limitation is the need to identify and purchase the target antigens, which can be accomplished in many cases at a relatively modest cost. Because these synthetic controls are of wholly defined composition and made with simple methods, they can be implemented in many laboratories with reproducible results. Their use may facilitate the objective, quantifiable evaluation of IHC staining results and allow greater intra- and inter-laboratory reproducibility.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-HER2/neu clone 4B5</td>
			<td>Ventana</td>
			<td>5278368001</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Wraps</td>
			<td>Leica</td>
			<td>3801090</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, ultra pure</td>
			<td>Cell Signaling Technology</td>
			<td>BSA #9998</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable base mold (15 mm x 15 mm)</td>
			<td>Fisher</td>
			<td>22-363-553</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable base mold 
			(24 mm x 24 mm)</td>
			<td>Fisher</td>
			<td>22-363-554</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable spatula</td>
			<td>VWR</td>
			<td>80081-188</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Thermomixer</td>
			<td>Eppendorf</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15686</td>
			<td></td>
		</tr>
		<tr>
			<td>ERBB2 / HER2 peptide</td>
			<td></td>
			<td></td>
			<td>UniProt P04626-1; a.a. 1240-55</td>
		</tr>
		<tr>
			<td>Leica Autostainer XL</td>
			<td>Leica</td>
			<td>ST5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stir Bar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoZoomer 2.0 HT whole slide imager</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>16004-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free microfuge tubes 1.5 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast paraffin</td>
			<td>Leica</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide parameter calculator</td>
			<td>Pep-Calc17</td>
			<td>https://www.pep-calc.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide suppliers</td>
			<td>ABclonal Science</td>
			<td></td>
			<td>Users should contact peptide vendors for details of mass, purity and cost.</td>
		</tr>
		<tr>
			<td></td>
			<td>Anaspec Peptide</td>
			<td></td>
			<td>Users should contact peptide vendors for details of mass, purity and cost.</td>
		</tr>
		<tr>
			<td></td>
			<td>CPC Scientific</td>
			<td></td>
			<td>Users should contact peptide vendors for details of mass, purity and cost.</td>
		</tr>
		<tr>
			<td></td>
			<td>New England Peptide</td>
			<td></td>
			<td>Users should contact peptide vendors for details of mass, purity and cost.</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline pH 7.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Alcohol</td>
			<td>Thermo Scientific</td>
			<td>9111</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Edge Razor</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus positively charged microscope slides</td>
			<td>Thermo Scientific</td>
			<td>6776214</td>
			<td></td>
		</tr>
		<tr>
			<td>TMA Tissue Grand Master</td>
			<td>3DHISTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>VWR</td>
			<td>89370-088</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthetic antigen controls for immunohistochemistry

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 &#176;C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities.
This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

Peptides should dissolve entirely in an appropriate solvent at room temperature to form an optically clear solution. If visible particulate material is still present after 30-60 min, it may be helpful to add additional volumes of the original solvent or an alternative solvent not exceeding the intended volume of the 5x peptide stock solution calculated in Table 1. Likewise, the combined peptide/BSA solution should remain translucent (Figure 1A).
Peptide/BSA gel samples should form an opaque rubbery mass after heating with 37% formaldehyde (Figure 1B-I). Minor fracturing of the gel samples may occur during removal from the microcentrifuge tubes (Figure 3A). These should not interfere with subsequent steps. Paraffin-embedded gel samples should section smoothly without tearing or chatter. Gels that show irregular tear-outs (Figure 3B) may benefit from less aggressive block facing and/or brief soaking in ice water or ammonia water. Areas of variably reduced signal in a watermark pattern may occur when the distribution of one or more staining reagents is uneven (Figure 3C). The focally increased macroscopic signal may be seen if there is reagent trapping under the gel sections at any stage of the staining process (Figure 3C). The use of positively charged glass slides and careful drying of slides after sectioning may reduce this artifact. Microscopic areas of the reduced signal may occur if there is an uneven distribution of antigen in the gel matrix or if variation in the local gel structure limits antibody-antigen interaction (Figure 3D). Incomplete peptide dissolution may result in scattered areas of focally intense signal in a low-intensity background (Figure 3E).
After reacting with an appropriate antibody, negative control BSA-only gel samples should show minimal signal (Figure 4A), optimally less than 2%-3% of the dynamic assay range. Very rarely, there may be significant antibody interaction with BSA gel material lacking any antigen that cannot be eliminated by changing reaction conditions. The signal intensity in an individual gel sample should be relatively uniform, with increasing signal in samples with increasing antigen concentrations (Figure 4A,B). The absolute signal intensity will vary depending on the antigen-antibody combination and conditions used for the IHC stain. Depending on the staining conditions, there may be a threshold antigen concentration below which the signal will be at&#160;background and an antigen concentration above which the signal will be saturated. In some assays, the signal above background may be visible in samples with as little as 2.5 E-8 M peptide2. In replicate staining runs, the signal intensity for sections cut from each gel sample should be reproducible from run to run. In this application of anti-HER2 antibody, the widely used MDA-175 and SK-BR-3 cell line controls were stained in the same experiment to compare to the peptide controls. The cell lines show the expected distribution and intensity of signal: &#62;10% of MDA-175 cells (HER2 1+) show faint (Figure 4C,D), incompletely circumferential membranous staining, and &#62;10% of SK-BR-3 cells (HER2 3+) (Figure 4E,F) show intense completely circumferential membranous staining.
Possible reasons for the absence of signal include: (1) the peptide sequence is not a functional target for the antibody; (2) the staining protocol is not optimized to detect the antigen concentrations present in the gels; (3) the wrong peptide sample was stained; (4) reagent or instrument error. Possible reasons for signal in samples where it is not expected include non-specific reactivity of primary antibody or detection reagents with the BSA gel or peptide samples that were mislabeled during preparation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62819/62819fig01.jpg" /> 
Figure 1: Peptide-BSA gel preparation. (A) 25% (w/v) BSA (without added peptide) in PBS, pH 7.2. (B) Gel formed by mixing equal volumes of 25% BSA and 37% formaldehyde solution and heating to 85 &#176;C for 10 min. (C-G) After cooling at room temperature for 5-10 min, the BSA gel can be removed from the microcentrifuge tube using a flexible disposable spatula. (H-I) The intact gel is sliced into discs ~5 mm thick in preparation for processing and embedding. Scale bars are 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62819/62819fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62819/62819fig02.jpg" /> 
Figure 2: Preparation of BSA gel samples for processing and embedding. (A-D) Preparation of a paraffin block containing a single disc of BSA gel. (A) The BSA-antigen gel is wrapped in biopsy tissue before processing. (B) After paraffin infiltration, the now-translucent gel disc is placed on the embedding center heated stage and unwrapped. (C) The single gel disc is placed in a 15 mm x 15 mm embedding mold containing liquid paraffin. (D) The completed paraffin block is ready for sectioning. (E-H) Preparation of a paraffin block containing multiple discs of BSA gel. As for Figures A-D above, the remaining gel samples are processed and embedded in one block to provide material for TMA construction. Scale bars are 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62819/62819fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62819/62819fig03.jpg" /> 
Figure 3: Gel preparation and staining artifacts. (A) Cores may show minor fracturing introduced during processing and/or embedding steps; focal darkening may reflect reagent trapping under the section. (B) Tearing out of gel material (seen in stained sections as irregular voids) may result from over- or under-soaking the block before sectioning. (C) Irregular watermark staining patterns may be caused by uneven reagent distribution at one or more staining steps. (D) Microscopic patterning of the BSA-peptide structure may be seen, particularly at higher peptide concentrations. (E) Incomplete peptide dissolution may produce a starry sky pattern with a focal signal in a low-intensity background. Scale bars are 200 &#181;m (A-C) or 20 &#181;m (D,E). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62819/62819fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62819/62819fig04.jpg" /> 
Figure 4: Stained ERBB2/HER2 peptide TMA with ERBB2/HER2 1+ and 3+ cell lines. (A) Replicate TMA cores (top and bottom rows) containing no peptide (left column) or ERBB2 peptide at concentrations ranging from 2.5 E-8 M to 2.5 E-4 M were stained with anti-HER2/neu clone 4B5 according to the manufacturer&#39;s recommendations. Cores with increasing peptide concentrations show a graded increase in signal. (B) The TMA cores shown in panel A were digitally scanned, and the average pixel intensity [0.01 x (100 - % transmittance) x 255] is plotted vs. peptide concentration. (C-F) Cell line positive controls containing ERBB2/HER2-expressing cell lines MDA-175 (HER2 1+, C,D) and SK-BR-3 (HER2 3+, E,F) were included in the TMA containing the BSA-peptide gels so that the intensity of signal in the cell lines and BSA-peptide gels could be compared on the same slide. Scale bars are 500 &#181;m (A), 250 &#181;m (C,E), and 20 &#181;m (D,F). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62819/62819fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A</td>
			<td colspan="2">B</td>
			<td>C</td>
			<td>D</td>
			<td>E</td>
			<td colspan="2">F</td>
			<td colspan="2">G</td>
			<td>H</td>
		</tr>
		<tr>
			<td>Peptide name</td>
			<td colspan="2">Peptide sequence</td>
			<td>MW (Da)</td>
			<td>Peptide purity (%)</td>
			<td>Peptide mass provided (mg)</td>
			<td colspan="2">Moles of peptide in sample (includes correction for purity)</td>
			<td colspan="2">Volume (microliters) of solvent in which to resuspend peptide to get 1.25 e-2 M stock</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>ERBB2 / HER2</td>
			<td colspan="2">Ac YGSGTPTAENP 
			EYLGLDVPVGSGC amide</td>
			<td>2424.6</td>
			<td>95.0</td>
			<td>20.0</td>
			<td colspan="2">7.84E-06</td>
			<td colspan="2">626.9</td>
			<td>DMF</td>
		</tr>
	</tbody>
</table>
Table 1: Calculations for the preparation of peptide stock.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A</td>
			<td>B</td>
			<td>C</td>
			<td>D</td>
			<td>E</td>
			<td>F</td>
			<td>G</td>
			<td>H</td>
			<td>I</td>
		</tr>
		<tr>
			<td>Tube</td>
			<td>5x Peptide stock</td>
			<td>1x Peptide stock</td>
			<td>Dilution 1</td>
			<td>Dilution 2</td>
			<td>Dilution 3</td>
			<td>Dilution 4</td>
			<td>Dilution 5</td>
			<td>Negative control</td>
		</tr>
		<tr>
			<td>[Peptide] stock (M)</td>
			<td>1.25*10-2</td>
			<td>2.5*10-3</td>
			<td>5*10-4</td>
			<td>5*10-5</td>
			<td>5*10-6</td>
			<td>5*10-7</td>
			<td>5*10-8</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Peptide solution 
			(in solvent)</td>
			<td>&#62;30 uL</td>
			<td>30 uL from 5x 
			(1.25E-2 M) 
			peptide stock</td>
			<td>140 uL from 1x 
			(2.5E-3 M) 
			&#160;peptide stock</td>
			<td>70 uL from 
			Dilution 1</td>
			<td>70 uL from 
			Dilution 2</td>
			<td>70 uL from 
			Dilution 3</td>
			<td>70 uL from 
			Dilution 4</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Solvent volume</td>
			<td></td>
			<td>120 uL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Final peptide 
			stock volume</td>
			<td></td>
			<td>150 uL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>31.3% BSA / PBS 
			volume</td>
			<td></td>
			<td></td>
			<td>560 uL 
			31.3% BSA</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25% BSA / PBS 
			volume</td>
			<td></td>
			<td></td>
			<td></td>
			<td>630 uL 
			25% BSA</td>
			<td>630 uL 
			25% BSA</td>
			<td>630 uL 
			25% BSA</td>
			<td>630 uL 
			25% BSA</td>
			<td>700 uL 
			25% BSA</td>
		</tr>
		<tr>
			<td>Volume of diluted peptide in BSA</td>
			<td></td>
			<td></td>
			<td>700 uL</td>
			<td>700 uL</td>
			<td>700 uL</td>
			<td>700 uL</td>
			<td>700 uL</td>
			<td>700 uL</td>
		</tr>
	</tbody>
</table>
Table 2: Calculations for the preparation of BSA-peptide dilutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A</td>
			<td>B</td>
			<td>C</td>
			<td>D</td>
			<td>E</td>
			<td>F</td>
			<td>G</td>
		</tr>
		<tr>
			<td>Tube</td>
			<td>Dilution 1</td>
			<td>Dilution 2</td>
			<td>Dilution 3</td>
			<td>Dilution 4</td>
			<td>Dilution 5</td>
			<td>Negative control</td>
		</tr>
		<tr>
			<td>Volume remaining of peptide in diluted BSA</td>
			<td>630 uL</td>
			<td>630 uL</td>
			<td>630 uL</td>
			<td>630 uL</td>
			<td>700 uL</td>
			<td>700 uL</td>
		</tr>
		<tr>
			<td>[Peptide] (M) in diluted BSA</td>
			<td>5*10-4</td>
			<td>5*10-5</td>
			<td>5*10-6</td>
			<td>5*10-7</td>
			<td>5*10-8</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Volume of 37% Formaldehyde to add</td>
			<td>630 uL</td>
			<td>630 uL</td>
			<td>630 uL</td>
			<td>630 uL</td>
			<td>700 uL</td>
			<td>700 uL</td>
		</tr>
		<tr>
			<td>Final [Peptide] (M) 
			in gel</td>
			<td>2.5*10-4</td>
			<td>2.5*10-5</td>
			<td>2.5*10-6</td>
			<td>2.5*10-7</td>
			<td>2.5*10-8</td>
			<td>None</td>
		</tr>
	</tbody>
</table>
Table 3: Calculations for the preparation of BSA-formaldehyde gels.

Watch this Scientific Journal Video about Synthetic Antigen Controls for Immunohistochemistry at JoVE.com

Synthetic Antigen Controls for Immunohistochemistry

This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires ...

synthetic-antigen-controls-for-immunohistochemistry

Nanyang Technological University

Research

JoVE Journal

Medicine

2.4K Views.  Genentech, Inc.. This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

Video: Synthetic Antigen Controls for Immunohistochemistry

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.

We have synthesized a novel analogue of pancratistatin with comparable anti-cancer activity as native pancratistatin; interestingly, combinatory treatment with tamoxifen yielded a drastic enhancement in apoptotic and autophagic induction by mitochondrial targeting with minimal effect on noncancerous fibroblasts. Thus, JCTH-4 in combination with tamoxifen could provide a safe anti-cancer therapy.

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce ...

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Intraoperative Gastroscopy for Tumor Localization in Laparoscopic Surgery for Gastric Adenocarcinoma

Determining resection margins for gastric cancer, which are not exposed to the serosal surface of the stomach, is the most important procedure during totally laparoscopic gastrectomy (TLG). The aim of this protocol is to introduce a procedure for intraoperative gastroscopy, in order to directly mark tumors during TLG for gastric cancer in the middle third of the stomach. Patients who were diagnosed with adenocarcinoma in the middle third of the stomach were enrolled in this case series. Before surgery, additional gastroscopy for tumor localization is not performed. Under general anesthesia, laparoscopic mobilization of the stomach is performed first. After the first portion of the duodenum is mobilized from the pancreas and clamped, the surgeon moves to the other side for the gastroscopic procedure. On the insertion of a gastroscope through the oral cavity into the stomach, 2 - 3 cc of indigo carmine is administered via an endoscopic injector into the gastric muscle layer at the proximal margin of the stomach. The location of stained serosa in the laparoscopic view is used to guide distal subtotal gastrectomy, however, total gastrectomy is performed if the tumor is too close to the esophagogastric junction. A specimen is sampled after distal gastrectomy to confirm sufficient length from resection margin to tumor before reconstruction. In our case series, all patients had tumor-free margins and required no additional resection. There was no morbidity related to the gastroscopic procedure, and the time required for the procedure has gradually decreased to about five minutes. Intraoperative gastroscopy for tumor localization is an accurate and tolerated method for gastric cancer patients undergoing totally laparoscopic distal gastrectomy.

In early gastric cancer, the aim of surgery is to precisely remove the distal stomach including the primary tumor. To do this, accurate localization of the tumor is crucial, especially in totally laparoscopic surgery. This protocol describes a procedure for intraoperative gastroscopy in totally laparoscopic subtotal gastrectomy.

Determining resection margins for gastric cancer, which are not exposed to the serosal surface of the stomach, is the most important procedure during ...

Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the extremely low delivery efficiency (&#60; 1%) to the olfactory mucosa that directly connects the brain. It is crucial to develop novel techniques to deliver neurological medications more effectively to the olfactory region. The objective of this study is to develop a numerical platform to simulate and improve intranasal olfactory drug delivery. A coupled image-CFD method was presented that synthetized the image-based model development, quality meshing, fluid simulation, and magnetic particle tracking. With this method, performances of three intranasal delivery protocols were numerically assessed and compared. Influences of breathing maneuvers, magnet layout, magnetic field strength, drug release position, and particle size on the olfactory dosage were also numerically studied.
From the simulations, we found that clinically significant olfactory dosage (up to 45%) were feasible using the combination of magnet layout and selective drug release. A 64 -fold higher delivery of dosage was predicted in the case with magnetophoretic guidance compared to the case without it. However, precise guidance of nasally inhaled aerosols to the olfactory region remains challenging due to the unstable nature of magnetophoresis, as well as the high sensitivity of olfactory dosage to patient-, device-, and particle-related factors.

This manuscript reviews the modeling and simulations of different protocols to deliver medications to the olfactory region in image-based nasal airway models. Multiple software modules are used to develop the anatomically accurate nose model, generate computational mesh, simulate nasal airflows, and predict particle deposition at the olfactory region.

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the ...

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

Synthetic Antigen Controls for Immunohistochemistry

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Chapters in this video

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Mouse Models for Graft Arteriosclerosis

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Intraoperative Gastroscopy for Tumor Localization in Laparoscopic Surgery for Gastric Adenocarcinoma

Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections