This work was supported by research grants to K.-M.H. from the National Cancer Center, Korea (1510121) and National Research Foundation, Korea (no. NRF-2015R1A2A2A04007432).

The study protocol was approved by the Institutional Review Board of National Cancer Center, Korea (NCCNCS-12-630).
1. Sample Preparation (30 min)
<ol>
	<li>For the culture of HeLa cells (CCL-2, ATCC), use 10 mL complete DMEM medium (10% fetal bovine serum, 1% pen-strep) in a 100 mm culture dish. To prepare serum-starved HeLa cells, incubate confluent HeLa cells in DMEM without fetal bovine serum for 48 hours, as described in a previous study11.</li>
	<li>To detach the cells, wash them with 2 mL phosphate-buffered saline (PBS), and add 2 mL 0.25% EDTA trypsin per 100 mm culture dish. Then, incubate for 2 - 3 min in a CO2 incubator at 37 &#176;C.</li>
	<li>Add 5 mL of complete DMEM medium to the culture dish to deactivate trypsin, and transfer the detached cells to a 15 mL tube. Then, centrifuge the cells at 300 x g for 10 min to form a pellet.</li>
	<li>Remove the supernatant, and wash the pellet twice with 2 mL cold PBS by centrifugation at 300 x g for 10 min.</li>
	<li>After removal of the supernatant, add 1 mL of 95% ethanol to the cell pellet, and mix the cell pellet with ethanol by vortexing. Keep the fixed cells on ice until the cell-block preparation.</li>
</ol>
2. Cell-block Preparation (1 h 30 min)
<ol>
	<li>For the preparation of frozen plasma aliquots, collect EDTA plasma from healthy donor blood and centrifuge at 13,000 x g for 10 min. Collect the supernatant plasma, and aliquot 200 - 400 &#181;L each into microfuge tubes. Store the aliquots at -80 &#176;C until use. When the frozen plasma aliquots are ready, retrieve them and thaw the plasma by incubation at 37 &#176;C for 5 min.</li>
	<li>Centrifuge the fixed cells at 700 x g for 10 min, then discard the supernatant.</li>
	<li>Mix the cell pellet with 2 drops (about 200 &#181;L) of the plasma, 2 drops of thromboplastin, and 2 drops of 0.025 M calcium chloride. Then, mix them by pipetting.</li>
	<li>Allow the mixture to form a cell clot at room temperature for 10 min, as shown in Figure 2A. Then, wash the cell clot with PBS two times by pouring and removing PBS by pipette. The representative white cell clot is shown in Figure 2B.</li>
	<li>Moisten a piece of filter paper with formalin, and wrap the cell clot with the pre-moistened filter paper. Then, place the clotted mixture with a pincette in a tissue cassette as shown in Figure 2C.</li>
	<li>Place the tissue cassette in a glass jar containing 50 mL of buffered formalin (10% formalin in PBS) overnight at 4 &#176;C for formalin fixation.</li>
</ol>
3. Tissue Processing and Paraffin Embedding (Overnight)
<ol>
	<li>Load the tissue cassette containing the formalin-fixed cell clot in a tissue processor overnight as described previously11. The tissue processing is for water removal and conditioning of fixed cells prior to paraffin embedding. For research without tissue processing, perform the tissue processing procedure as shown in Table 1.</li>
	<li>Turn on the heated embedding station, and 60 minutes later, check that there is molten paraffin in the metal mold therein (Figure 2D).</li>
	<li>Take the formed cell clot from the tissue cassette and place it in the molten paraffin within the metal mold.</li>
	<li>Place the new tissue cassette without the lid into the metal mold containing the cell clot in the middle of the molten paraffin, and pour more molten paraffin into the tissue cassette and onto the metal mold. Then, let the paraffin solidify in the cold plate for 30 - 60 seconds.</li>
	<li>Separate the tissue cassette from the metal mold. Then, the paraffin cell block is ready for immunocytochemistry. The embedded cell clot in the paraffin-embedded cell block is indicated by the arrow in Figure 2E.</li>
</ol>
4. Preparation of Slides for Immunocytochemistry (1 h)
<ol>
	<li>Check the area for the cell clot in the paraffin cell block, and cut the cell block into slices with thicknesses of 3 - 4 &#181;m using a microtome. Put the paraffin sections on silane-coated glass slides, as shown in Figure 2F.</li>
	<li>Place the section slides in a 37 &#176;C oven for 30 min to make the sections adhere to the slides.</li>
</ol>
5. Immunocytochemistry of Cell Blocks (6 h)
NOTE: For immunocytochemical staining on cell-block sections, various kits can be used (see Table of Materials). All such kits have various sensitivities and specificities depending on their modifications.
<ol>
	<li>Incubate the sections in 50 mL of xylene for 4 min to de-paraffinize.</li>
	<li>Incubate the slides in 100% ethanol for 2 min for dehydration, and two times in 95% ethanol, and in 80% ethanol for 2 min. Then, remove the ethanol in running water for 10 min.</li>
	<li>For antigen retrieval, place the slides in a jar containing 40 mL Tris-EDTA retrieval buffer, pH 9.0, and boil in a cooker for 30 min.</li>
	<li>Wash the slides under running water, and incubate them in 95% ethanol for 10 min at 4 &#176;C. Then, mark the cell-staining area on the slides with a Pap pen for easy identification.</li>
	<li>Wash the slides in Tris-buffered saline containing 0.2% Tween 20 (TBS-T), and incubate in the hydrogen peroxide block (see Table of Materials) at room temperature for 15 min to remove any remnant peroxidase activity. Then, wash the slides in TBS-T three times for 2 min each.</li>
	<li>Prepare diluted antibody solution by diluting primary antibody in the protein block (see Table of Materials). For example, the CKAP2 primary antibody can be diluted 1:100, and the Ki-67 antibody 1:500. Then, incubate the slides in 100 &#181;L of diluted antibody solution for 1 h.</li>
	<li>After washing in TBS-T five times for 2 min each, incubate the slides in the primary antibody enhancer in the kit for 15 min at room temperature in the dark.</li>
	<li>After the washing in TBS-T four times for 2 min each, add 2 drops of HRP polymer (a secondary antibody labeled with horseradish peroxidase), and incubate the slides at room temperature for 30 min.</li>
	<li>After washing in TBS-T five times for 2 min each, add 100 &#181;L of diaminobenzidine (DAB) solution (see Table of Materials), and incubate the slides for 3 min.</li>
	<li>After washing in TBS-T two times, add 100 &#181;L of Hematoxylin solution (the mixture of 100 &#181;L of Hematoxylin and 600 &#181;L of distilled water), and incubate the slides for 1 min.</li>
	<li>After washing the slides in TBS-T once, dehydrate by incubating in 95% ethanol for 2 min, dipping in 95% ethanol once, and dipping in 100% ethanol two times. Then, incubate the slides in 40 mL xylene in a glass jar for 5 min.</li>
	<li>When the slides are dry from xylene, mount the respective coverslips.</li>
	<li>Observe the staining patterns using light microscopy.</li>
</ol>

<ol>
	<li>Bhattacharyya, D., Hammond, A. T., Glick, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-quality+immunofluorescence+of+cultured+cells.">High-quality immunofluorescence of cultured cells.</a> Methods Mol Biol. 619, 403-410 (2010).</li><li>Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy+-+Avoiding+the+pit+falls.">Fluorescence microscopy - Avoiding the pit falls.</a> J Cell Sci. 120, 1703-1705 (2007).</li><li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+Techniques.">Immunofluorescence Techniques.</a> J Invest Dermatol. 133, e4 (2013).</li><li>Kulkarni, M. B., Desai, S. B., Ajit, D., Chinoy, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+the+thromboplastin-plasma+cell-block+technique+for+fine-needle+aspiration+and+serous+effusions.">The utility of the thromboplastin-plasma cell-block technique for fine-needle aspiration and serous effusions.</a> Diagn Cytopathol. 37 (2), 86-90 (2009).</li><li>Shivakumarswamy, U., Arakeri, S. U., Karigowdar, M. H., Yelikar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+utility+of+the+cell+block+method+versus+the+conventional+smear+study+in+pleural+fluid+cytology.">Diagnostic utility of the cell block method versus the conventional smear study in pleural fluid cytology.</a> J Cytol. 29 (1), 11-15 (2012).</li><li>Nathan, N. A., Narayan, E., Smith, M. M., Horn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+block+cytology:+Improved+preparation+and+its+efficacy+in+diagnostic+cytology.">Cell block cytology: Improved preparation and its efficacy in diagnostic cytology.</a> Am J Clin Pathol. 114 (4), 599-606 (2000).</li><li>Keyhani-Rofagha, S., Vesey-Shecket, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value,+feasibility,+and+validity+of+preparing+cell+blocks+from+fluid-based+gynecologic+cytology+specimens.">Diagnostic value, feasibility, and validity of preparing cell blocks from fluid-based gynecologic cytology specimens.</a> Cancer. 96, 204-209 (2002).</li><li>Young, N. A., Naryshkin, S., Katz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value+of+electron+microscopy+on+paraffin-embedded+cytologic+material.">Diagnostic value of electron microscopy on paraffin-embedded cytologic material.</a> Diagn Cytopathol. 9, 282-290 (1993).</li><li>Jin, Y., Murakumo, Y., Ueno, K., Hashimoto, M., Watanabe, T., Shimoyama, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mouse+cytoskeleton-associated+protein,+CKAP2,+with+microtubule-stabilizing+properties.">Identification of a mouse cytoskeleton-associated protein, CKAP2, with microtubule-stabilizing properties.</a> Cancer Sci. 95 (10), 815-821 (2004).</li><li>Hong, K. U., Choi, Y. -. B., Lee, J. -. H., Kim, H. -. J., Kwon, H. -. R., Seong, Y. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+phosphorylation+of+tumor+associated+microtubule+associated+protein+(TMAP)/cytoskeleton+associated+protein+2+(CKAP2)+at+Thr-596+during+early+phases+of+mitosis.">Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.</a> Exp Mole Med. 40, 377-386 (2008).</li><li>Sim, S. H., Bae, C. D., Kwon, Y., Hwang, H. -. L., Poojan, S., Hong, H. -. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CKAP2+(cytoskeleton-associated+protein2)+is+a+new+prognostic+marker+in+HER2-negative+luminal+type+breast+cancer.">CKAP2 (cytoskeleton-associated protein2) is a new prognostic marker in HER2-negative luminal type breast cancer.</a> PLoS ONE. 12 (8), e0182107 (2017).</li><li>Inwald, E. C., Klinkhammer-Schalke, M., Hofstadter, F., Zeman, F., Koller, M., Gerstenhauer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki-67+is+a+prognostic+parameter+in+breast+cancer+patients:+results+of+a+large+population-based+cohort+of+a+cancer+registry.">Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry.</a> Breast Cancer Res Treat. 139 (2), 539-552 (2013).</li><li>Katikireddy, K. R., O'Sullivan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+immunofluorescence+procedures+for+protein+analysis.">Immunohistochemical and immunofluorescence procedures for protein analysis.</a> Methods Mol Biol. 784, 155-167 (2011).</li><li>Beutner, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescent+staining:+The+fluorescent+antibody+method.">Immunofluorescent staining: The fluorescent antibody method.</a> Microbiol Mol Biol Rev. 25 (1), 49-76 (1961).</li></ol>

All authors have declared no conflicts of interest.

Immunofluorescent staining on fixed cultured cells is currently the method of choice for determination of protein expression level in cells while preserving morphological information1. However, immunocytochemistry on paraffin-embedded cell blocks can be an excellent alternative. The detailed procedures for the preparation of paraffin-embedded cell blocks and immunocytochemistry have been described in this protocol, and we hope it can facilitate the application of immunocytochemistry in cell studies.
Immunocytochemistry has several advantages over immunofluorescent staining. Immunofluorescent staining for cells usually requires freshly cultured cells, but paraffin cell blocks can be kept at room temperature for several years13. Additionally, immunocytochemistry on cell blocks can explore intracellular expression patterns by employing the same antibody used in routine immunohistochemistry on human tissues4. Furthermore, it can explore the changes in protein levels or posttranslational modifications either by pre-incubating cells with various drugs or under various culture conditions11.
In contrast to the advantages of immunocytochemical staining, the preparation of paraffin-embedded cell blocks takes time and is costly14. Also, most research laboratories lack experience in this technique, and technical errors under such circumstances are common. The most common errors are poor preservation of cell morphology and poor or irregular immunocytochemical staining on the paraffin cell block sections. These and most others can be avoided by making cell blocks under the best cell conditions and using enough solution to form cell clots.
As a demonstration of the present protocol, cell blocks were prepared for HeLa cells, and immunocytochemical staining was performed for two proliferation markers, CKAP2 and Ki-67, as previously reported11. For immunocytochemistry, the cells were manipulated by incubation in media with and without fetal bovine serum, and the effect of serum starvation could be observed. These prepared paraffin-embedded cell blocks can be employed for a large number of antibodies, because many slides can be prepared from a cell block using only a 4 - 5 &#181;m-thick cell-block section per slide. Therefore, expression patterns corresponding to two different conditions can be evaluated with several different antibodies. The immunostaining patterns for CKAP2 and Ki-67 in cancer tissues have already been reported9,10,11,12, and the immunocytochemical staining results could be easily evaluated, because the staining patterns were quite similar to those from immunohistochemistry.
In conclusion, immunocytochemical staining on paraffin cell blocks can be an excellent alternative to immunofluorescent staining; moreover, it can be easily and reliably employed in basic research for expression profiling in cell lines while preserving morphological information.

In most laboratories, paraffin-embedded cell blocks are not commonly used. Rather, fixed cultured cells, not paraffin-embedded cells, are employed in subcellular localization studies. For those fixed cultured cells, fluorescence instead of chromogen has been used. Therefore, immunofluorescent staining is currently the method of choice for determination of protein expression levels in research employing cell cultures1. Slides prepared for immunofluorescent staining, however, can be observed only under immunofluorescent microscopy, which might show images quite different from those indicated under plane microscopy2. Additionally, preservation of slides for immunofluorescent staining requires protection from bright light, and fluorescent signals become weaker with repeated exposure for imaging due to the loss of the fluorescent signal3. Results from immunocytochemistry on paraffin-embedded cell blocks are quite similar to those from immunohistochemistry on paraffin-embedded tissues4, and they can easily be translated into clinical information. Therefore, immunocytochemistry can be an excellent alternative. However, cell-block preparation has not been popular in basic research laboratories. In this protocol, then, cell-block preparation and immunocytochemical staining are provided to promote the use of this method in the field of cell-culture studies.
Cell-block preparation and immunocytochemistry are not unique methods, and they have already been applied from clinical diagnosis to basic research4,5. Although various cell-block preparation methods have been reported4,6 the thromboplastin-plasma method is simple, cost-effective, and readily adaptable. Therefore, in the protocol presented in this paper, the thromboplastin-plasma method4,5,6,7,8 is used for preparation of paraffin-embedded cell blocks.
In the present study, the detailed procedures for the preparation of thromboplastin-plasma cell blocks and the immunocytochemistry method employing two proliferation markers were demonstrated. One marker is cytoskeleton-associated protein 2 (CKAP2), which has recently been reported as a mitotic marker9,10,11; the other one is Ki-67, which is the best-known proliferation marker12. The representative scheme is shown in Figure 1.

<table><tbody><tr><td>HeLa Cells</td><td>ATCC</td><td>HeLa (ATCC&amp;nbsp;CCL-2)</td><td></td></tr><tr><td>Ki-67 Antibody</td><td>Thermo Fisher Scientific</td><td>RM-9106-S1</td><td></td></tr><tr><td>Paraffin</td><td>Leica</td><td>39601006</td><td></td></tr><tr><td>Xylene</td><td>Fisher SCientific</td><td>1330-20-7,100-41-4</td><td></td></tr><tr><td>Ethenol</td><td>GD Chem</td><td>DJ16016</td><td></td></tr><tr><td>Hematoxylin</td><td>Agilent</td><td>10118581</td><td></td></tr><tr><td>DAB Quanto Kit</td><td>Thermo Fisher Scientific</td><td>TA-125-QHDX, QHCX 170405</td><td></td></tr><tr><td>Ultravision LP detection Kit</td><td>Thermo Fisher Scientific</td><td>PBQ141209, LPB141209, LPH141210</td><td>Kit for immunocytochemistry; contains protein block</td></tr><tr><td>TintoRetriever Pressure Cooker</td><td>Bio SB Corporation</td><td>BSB 7008</td><td></td></tr><tr><td>Tris-buffered saline</td><td>iNtRON Biotechnology</td><td>IBS-BT008</td><td></td></tr><tr><td>Tween 20</td><td>USB Corporation</td><td>115106</td><td></td></tr><tr><td>Thromboplastin</td><td>Neoplastin Cl Plus</td><td>NC0591432</td><td></td></tr><tr><td>Tris-EDTA retrival Buffer</td><td>Dignostic Biosystem</td><td>E625-A</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Fetal&amp;nbsp;bovine serum (FBS)</td><td>Hyclone Laboratories Inc</td><td>SH30910.03</td><td></td></tr><tr><td>DMEM</td><td>Hyclone Laboratories Inc</td><td>SH30243.01</td><td></td></tr><tr><td>Antibiotic-Antimycotic, 100X</td><td>Thermo Fisher Scientific</td><td>15240062</td><td></td></tr><tr><td>Ultra vision peroxide block H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Thermo Fisher Scientific</td><td>02Q141212</td><td></td></tr><tr><td>Mounting Medium</td><td>Thermo Fisher Scientific</td><td>363313</td><td></td></tr><tr><td>Pap-Pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Filter Paper</td><td>GE Healthcare Life Sciences</td><td>1001-0155</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>21115-100ml</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>PAA Laboratories</td><td>H21-002</td><td></td></tr><tr><td>Formalin</td><td>Daejung</td><td>50-00-0</td><td></td></tr><tr><td>15 ml tube</td><td>SPL Life Sciences</td><td>50015</td><td></td></tr><tr><td>tissue processing cassette</td><td>Simport</td><td>M492-5</td><td></td></tr><tr><td>100 mm culture dishes</td><td>BD Biosciences</td><td>08-772E</td><td></td></tr><tr><td>Glass Slide</td><td>Muto Pure Chemicals</td><td>140712</td><td></td></tr><tr><td>Cover Glass</td><td>Marienfeld</td><td>2262817</td><td></td></tr><tr><td>Glass Zar</td><td>Hyunil Lab-Mate</td><td>HIP-1027</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Class II Laminar Flow Hood</td><td>Thermo Fisher Scientific</td><td>1300 series A2</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Fisher Scientific</td><td>Series A2</td><td></td></tr><tr><td>Tissue Processor</td><td>Leica BioSystem</td><td>TP1020</td><td></td></tr><tr><td>Microtome</td><td>Thermo Fisher Scientific</td><td>HM340E</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>CX-21</td><td></td></tr><tr><td>Paraffin embedding station</td><td>Thermo Fisher Scientific</td><td>EC 350-1, EC 350-2</td><td></td></tr><tr><td>Tissue Section Bath (Round)</td><td>CellPath</td><td>HCP-JAW-0100-00AEU</td><td></td></tr><tr><td>Fume Hood</td><td>Hanyang Scientific Equipment Co. Ltd.</td><td>FH-150</td><td></td></tr></tbody></table>

determination of protein expression level in cultured cells by immunocytochemistry on paraffin-embedded cell blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

In a hematoxylin-and-eosin-stained slide from the paraffin-embedded cell block (Figure 3A,B), most of the nuclei and cytoplasm of the cells are intact, suggesting that the morphological preservation is excellent with the current protocol (Figure 3A). In the immunocytochemical staining, positive CKAP2 staining was observed in condensed chromatin, mitotic spindle, and cytoplasm (Figure 4), as previously reported10. Ki-67 staining was observed in the cell nuclei, as expected (Figure 4). Only the cells with CKAP2 staining in condensed chromatin (see the arrows in Figure 4A,B) were mitotic cells. Many CKAP2-positive cells were shown in the highly mitotic HeLa cells that had been prepared after an incubation in complete medium&#160;(Figure 4A). In comparison, there were few CKAP2-positive cells in the serum-starved HeLa cells (Figure 4B). Most of the highly mitotic HeLa cells were Ki-67 positive (Figure 4C). Contrastingly, the Ki-67-positive rate in the serum-starved HeLa cells remained as high as ~50% (Figure 4D). These results are quite comparable to those of a previous report11, which suggests that CKAP2 is a more reliable proliferation marker in cancer cells than is Ki-67.
In poorly prepared cell blocks, the nuclei are separated from the cytoplasm, and there is also, resultantly, poor morphologic preservation. Longer-than-overnight incubation of fixed cells in a refrigerator might cause such poor results. Another important problem is that cells are not stained well by immunocytochemistry, notwithstanding the excellent morphology. This problem arises more often when the cell clot is small. Typically, the staining intensity is irregular as shown in Figure 3B; but when the cell clot is larger, there is much less chance of irregular staining. Therefore, in this protocol, we increased the volumes of plasma, thromboplastin, and calcium chloride in order to form a large cell clot.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57369/57369fig1.jpg" /> 
Figure 1: Cell-block preparation scheme. <a href="/files/ftp_upload/57369/57369fig1large.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/57369/57369fig2.jpg" /> 
Figure 2: Illustration of paraffin cell-block preparation. (A) Thromboplastin-plasma cell clot in tube. (B) TP Cell clot after PBS washing. (C) Cell clot on moistened filter paper. (D) Tissue-embedding station with molten wax. Metal mold (arrow) holds molten wax for solidification. (E) Paraffin-embedded cell clot (arrow) embedded in paraffin or paraffin-embedded cell block. (F) Thin paraffin section on the middle part of a coated slide. <a href="/files/ftp_upload/57369/57369fig2large.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/57369/57369fig3.jpg" /> 
Figure 3: Cell-block preparation and confirmation of quality by hematoxylin and eosin and immunostaining. (A) Hematoxylin-and-eosin-stained image of HeLa cells in a paraffin-embedded cell-block section. (B) Irregular staining of Ki-67 in immunocytochemistry on a poorly prepared paraffin-embedded cell-block section. Scale bars are (A) 100 and (B) 200 &#956;m. <a href="/files/ftp_upload/57369/57369fig3large.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/57369/57369fig4.jpg" /> 
Figure 4: Immunocytochemical staining on paraffin-embedded cell block for HeLa cells. (A) CKAP2 staining under highly mitotic conditions. (B) CKAP2 staining under serum-starved conditions. (C) Ki-67 staining under highly mitotic conditions. (D) Ki-67 staining under serum-starved conditions. 100 &#956;m scale bars are shown. The arrowheads indicate CKAP2-positive cells. <a href="/files/ftp_upload/57369/57369fig4large.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>Procedure</td>
			<td>Steps</td>
			<td>Solution</td>
			<td>Time/Temperature</td>
		</tr>
		<tr>
			<td rowspan="4">Dehydration</td>
			<td>1</td>
			<td>70% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td>2</td>
			<td>80% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td>3</td>
			<td>95% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td>4</td>
			<td>100% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td rowspan="4">Clearing</td>
			<td>1</td>
			<td>Xylene</td>
			<td>60 min/4 &#176;C</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Xylene</td>
			<td>10 min/RT</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Xylene</td>
			<td>10 min/RT</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Xylene</td>
			<td>10 min/RT</td>
		</tr>
		<tr>
			<td>*RT, room temperature</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Tissue processing procedure.

Watch this Scientific Journal Video about Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks at JoVE.com

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

determination-protein-expression-level-cultured-cells

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.4K Views.  National Cancer Center. Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Video: Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Medicine

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Biology

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Isolation of Stably Transfected Melanoma Cell Clones

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated cell transfection as it can lead to a heterogeneous cell population and variable transfection efficiency, affecting reproducibility. Moreover, they are preferable for large-scale analyses. The generation of stable cell clones is useful for a wide range of applications, such as research on gene functions and recombinant protein production. There are a few methods to obtain a homogenous cell population upon initial transient transfection. Here, we describe the isolation of single cell clones with glass cylinders. Although this method has been known for some time, there are a few crucial steps, and neglecting them may lead to failure. We have successfully used this method to obtain clones stably overexpressing a protein of interest (POI) or with knockout of a gene of interest (GOI). We describe preparation steps such as the optimization of selecting drug concentrations, preparation of glass cylinders, and validation of whether the obtained clones have the desired change in the expression of the GOI by PCR, western blot analysis, immunostaining, or gDNA sequencing (depending on the type of derived clones). We also discuss the phenotypic heterogeneity of well-established cell lines as this might be an issue in obtaining stable cell clones.

A protocol is described for isolating stably transfected melanoma cell clones using glass cylinders. We focus on practical advice that may be decisive for the success of the entire procedure.

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

Standardized Processing for Formalin-Fixed, Paraffin-Embedded Cell Pellet Immunohistochemistry Controls

Positive and negative controls with known expression of target proteins are essential for the development of immunohistochemistry (IHC) assays. While tissue controls are beneficial for well-characterized proteins with defined tissue and cellular expression patterns, they are less suitable for the initial development of IHC assays for novel, poorly characterized, or ubiquitously expressed proteins. Alternatively, due to their standardized nature, cell pellets, including cancer cell lines with defined protein or transcript expression levels (e.g., high, medium, and low expression), transfected over-expressing cell lines, or cell lines with genes deleted through cell engineering technologies like CRISPR, can serve as valuable controls, especially for the initial antibody characterization and selection. In order for these cell pellets to be used in the development of IHC assays for formalin-fixed, paraffin-embedded tissues, they need to be processed and embedded in a manner that recapitulates the procedures used for tissue processing. This protocol describes a process for creating and processing formalin-fixed, paraffin-embedded cell pellet controls that can be used for IHC method developments.

Presented here is a protocol for generating formalin-fixed, paraffin-embedded cell pellet controls for immunohistochemistry.

Positive and negative controls with known expression of target proteins are essential for the development of immunohistochemistry (IHC) assays. While ...

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

Source: Michael S. Lee1 and Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. The first published use of IHC was in 1941 when Albert Coons used the technique to visualize the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus (1). The name, immunohistochemistry, is derived from the roots &#34;immuno-,&#34; in reference to antibodies, and &#34;histo-,&#34; in reference to the tissue sections used in IHC. The root &#34;cyto-&#34; in immunocytochemistry highlights the key difference between ICC and IHC. Whereas IHC uses sections of whole tissue, ICC uses cells that have been isolated from tissue or grown in culture. The difference in samples used means sample preparation technically differs between IHC and ICC, but otherwise the protocols for ICC and IHC are identical and one will find the terms are frequently used interchangeably.
In both IHC and ICC, antibodies with either chemical or fluorescent tags, such as peroxidase or rhodamine, respectively, are used to visualize the distribution of any antigen of interest through specific binding of the tagged antibody to the antigen. In the case of IHC, thin slices of tissue are immobilized on a slide to maintain the structure of the tissue before being stained, allowing the visualization of antigens in the context of whole tissues (Figure 1). In the case of ICC, cells are distributed evenly on a slide before being stained, allowing the visualization of antigen distribution within individual cells but not within the structure of any specific tissue. Due to the similarities between the two protocols, this protocol will focus on IHC to address the additional complexities of sample preparation involved in IHC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10498/10498fig1.jpg" /> 
Figure 1: Outline of IHC Protocol. Visual outline of an IHC protocol for paraffin-embedded tissue dissected from a mouse. This protocol uses a biotinylated secondary antibody and strepavidin-HRP to visualize the location of antibody binding. Other options, such as fluorescently tagged antibodies, are also possible. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10498/10498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The first major decision when performing IHC is how to prepare the tissue sections in order to maintain the structure of the tissue throughout the staining process. The two main choices are formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. There is no simple answer as to which method to use as it depends on what downstream analysis will be conducted. Formalin-fixation of paraffin embedded tissues is generally thought to better preserve tissue morphology for optimal imaging while freezing fresh tissue can preserve protein function for subsequent assays outside of IHC. In addition, fresh frozen tissue sections have been shown to be more suitable for gene expression analysis (2). A third consideration is whether or not the antibodies for your antigen of interest are suitable for either fixed or frozen tissue sections, as some antibodies have only been optimized for a specific type of section and may not work for others. Finally, one also needs to determine how long they need to store the tissue sections, as fresh frozen samples must be kept at -80&#176;C and may not last beyond one year while fixed sections can be stored for much longer at room temperature. These are a few of the major considerations for determining whether to use formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. Ultimately, if one has enough tissue, it may be best just to have some of both.
In this experiment, we set out to determine if cyclin D1 expression was increased in enlarged spleens from a spontaneous mouse model of lymphoma development. Splenic tissue samples were first isolated from either wild-type mice, transgenic mice that do not have lymphoma, or transgenic mice that have spontaneously developed lymphoma. The spleen tissue samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, stained using a mouse anti-cyclin D1 primary antibody followed by a horse anti-mouse secondary antibody, and developed using 3,3-diaminobenzidine (DAB). The sections were then counterstained in Harris Hematoxylin Solution and then the sections were imaged at 20X magnification.
Reagents
Paraffin-Embedded Sections
<ol type="a">
	<li>4% Paraformaldehyde (PFA)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100%, 95%, 80%, 75%, and 50%). Can be diluted from 100% stock using double distilled water (ddH2O)</li>
	<li>Xylene</li>
	<li>IHC compatible glass slide to ensure tissue section remains attached throughout the entire procedure. IHC compatible glass slides have a specialized coating and are readily available from multiple retailers. If performing ICC, use a chambered slide. Chambered slides allow for cells to be seeded in the chambers and placed in the incubator until cells attach to the slide and reach proper confluency, at which point the chambers can be removed and staining can proceed in the same manner as IHC.</li>
	<li>Paraffin</li>
	<li>0.3% Hydrogen peroxide (H2O2)/methanol: To prepare, add 1 mL 30% H2O2 to 99 mL methanol. Store at -20&#176;C</li>
	<li>Antigen retrieval buffer: IHC citrate buffer pH 6.0</li>
</ol>
Fresh Frozen Sections
<ol type="a">
	<li>Optimal cutting temperature (OCT) embedding compound</li>
	<li>Optimal fixative: 4% PFA or acetone that has been cooled to -20&#176;C</li>
</ol>
Staining
<ol type="a">
	<li>Blocking buffer: Should be determined by user. One example is horse serum diluted in 1X PBS</li>
	<li>Diluted primary antibody: see manufacturer specifications</li>
	<li>Diluted biotinylated secondary antibody: see manufacturer specifications</li>
	<li>Diluted avidin-Horseradish Peroxidase (HRP): Only for peroxidase visualization. See manufacturer specifications.</li>
	<li>DAB or another compatible substrate</li>
	<li>Counterstain (optional)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100% and 95%)</li>
	<li>Xylene</li>
	<li>Organo/Limonene Mount</li>
</ol>

Immunohistochemistry &amp; Immunocytochemistry: Tissue Imaging

Source: Michael S. Lee1 and Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and ...