We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).

All the animal-involved procedures were performed in accordance with the University of Nottingham&#39;s ethical review board.
1. Selecting Suitable Tissue Preparation for Immunostaining
<ol>
	<li>Generate paraffin-embedded section of wild-type CD1 mouse embryos and adult brain tissues as described previously25. Use brain tissue sections fixed with either 4% formaldehyde (FA) or 4% paraformaldehyde (PFA) for immunostaining method25. 
	NOTE: The use of paraffin embedded tissue sections requires de-waxing prior to antibody labelling. Since the treatment with 2 - 4 M hydrochloric acid (HCl) employed in the protocol for DNA denaturing is not compatible with most antigen retrieval strategies, we recommend the use of either cryo- or microtome sections for co-detection of 5mC oxidation derivatives with protein markers.</li>
</ol>
2. Dewaxing Paraffin Embedded Tissue Sections
<ol>
	<li>In a running class II safety cabinet, wash the paraffin embedded tissue sections in a Coplin jar filled with xylene, 2 times for 10 min each at RT. 
	NOTE: It is important to use fresh xylene as incomplete paraffin removal can lead to inconsistent staining patterns.</li>
	<li>After de-waxing, rapidly rehydrate the tissue sections by washing consecutively in 95, 75, and 50% ethanol for 10 min each at RT.</li>
</ol>
3. Fixation and Permeabilization of Cryo- and Microtome Sections
<ol>
	<li>Fix the rehydrated tissue sections by placing them in either ice cold 4% PFA or 4% FA for 15 min at RT.</li>
	<li>Remove excess fixative by washing the sections in PBS (Phosphate buffer saline) for 5 min at RT.</li>
	<li>Permeabilize the tissue sections by placing in a Coplin jar filled with PBX (0.5% Triton X-100 in PBS) for 30 min at RT. Remove excess PBX by washing the sections shortly in PBT (0.01% Tween 20 in PBS).</li>
</ol>
4. Immunostaining for Oxi-5mC Derivatives
<ol>
	<li>Place the permeabilized-sections in 2 N HCl for 60 min at RT for depurination of the DNA. 
	NOTE: Although higher concentrations of HCl (e.g., 4 N) lead to a more efficient DNA denaturing, they are not compatible for co-detection of oxi-mCs with DAPI.</li>
	<li>Place the sections in 10 mM Tris-HCl (pH 8.5) for 30 min at RT to neutralize the HCl. Alternatively, wash the sections three times for 5 mins each in PBS. Incubate the sections in PBT for 5 min at RT.</li>
	<li>Carefully remove the liquid from the area surrounding the tissue section without letting the tissue section to dry completely at any step by gently shaking the slide. Use a hydrophobic barrier pen to encircle the section without touching the section. 
	NOTE: The hydrophobic barrier pen reduces the volume of antibody required to stain the tissue and can allow multiple sections to be stained with different antibodies on the same slide.</li>
	<li>Incubate sections in 100 &#181;l of blocking solution (10% bovine serum albumin in PBS) for 1 hr at RT in a humid chamber. 
	NOTE: Skipping this step would not affect the efficiency of staining the modified cytosine bases.</li>
	<li>Incubate the tissue sections in 100 &#181;l of a 1:5,000 dilution of mouse monoclonal anti-5hmC and a 1:1,000 dilution of rabbit polyclonal anti-5caC primary antibodies in blocking solution for 1 hr at RT in a humid chamber. Alternatively, perform the incubation overnight at 4 &#176;C if needed. 
	NOTE: Sections processed without primary antibodies can serve as suitable negative controls for the staining procedure. The 12.5 days post coitum murine embryonic brain sections enriched in 5caC, 5fC and 5hmC 20 can be used as positive control.</li>
	<li>Remove excess antibodies by washing the sections in a Coplin jar filled with PBT three times for 5 min each in a Coplin jar at RT. 
	NOTE: Increasing the volume of washing solutions can reduce background staining.</li>
	<li>Remove excess PBT and, if necessary, encircle the sections again with the hydrophobic barrier pen as PBT contain detergent that can weaken the hydrophobic barrier.</li>
	<li>Make a 1:400 dilution of goat anti-rabbit HRP-conjugated antibody and a 1:400 dilution of donkey anti-mouse 555-conjugated antibody in blocking solution.</li>
	<li>Incubate the tissue sections in 100 &#181;l of secondary antibody mixture from step 4.9 for 1 hr at RT in a humid chamber.</li>
	<li>Wash the tissue sections in a Coplin jar filled with PBT three times for 5 min each at RT.</li>
	<li>Place the tissue sections in 100 &#181;l of a 1:200 dilution of tyramide in the tyramide signal amplification buffer for 2 min at RT.</li>
	<li>Immediately, remove excess tyramide solution by washing the slides three times for 5 min each in PBT.</li>
	<li>Carefully remove excess PBT and immediately cover the sections with a drop of a Mounting Medium (see Materials List).</li>
	<li>Gently place a coverslip on the tissue sections and immediately seal the coverslip with nail polish.</li>
	<li>Store the tissue sections at 4 &#176;C for several hr before microscopic examination. Examine under a fluorescent microscope at 405, 488 and/or 555 nm (10 - 40X magnification).</li>
</ol>

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The authors declare no conflict of interest.

Although the reported low abundance of 5mC oxidation derivatives, 5fC and 5caC in some tissues would present significant limitations for a standard immunochemistry protocol, the incorporation of peroxidase-conjugated secondary antibodies allowed the detection of these cytosine modifications in fixed tissues and cells (Figure 2). However, the optimal incubation time with the tyramide solution should be optimized experimentally for each individual batch of tyramide signal amplification kit where a linear relationship between signal intensity and duration of tyramide based signal amplification is observed, for details refer to Almeida et al. 201226. In addition, the signal/background ratio can be significantly enhanced by carrying the washes in a Coplin jar to allow efficient removal of excess antibodies. Following incubation with tyramide solution, it is important to immediately stop the reaction by washing in PBT solution to decrease background staining. It is critical not to allow the sections to dry at any point during the procedure.
The efficiency of DNA depurination can be improved by carrying out the depurination reaction at 37 &#176;C. While using 4 N HCl instead of 2 N enhances the staining of the modified forms of 5mC using 4 N HCl for DNA depurination would not permit co-staining with DAPI, as it interacts exclusively with double stranded DNA.
Although this technique can provide robust semi-quantitative assessment of the modified forms of cytosine bases where detectable, it cannot be used for evaluating the absolute levels of 5mC or its oxidation derivatives. Therefore, we recommend the use of other complementary but quantitative approaches16, 19 -24. Since the discovery of 5mC oxidation derivatives in mammalian genomes, several approaches have been developed to study their biological roles16, 19-24. Although these approaches can be valuable in determining the absolute levels of 5mC oxidation derivatives, they do not provide information concerning their spatial distribution20, 26. We have successfully used the method described here to map the spatial distribution and localization of 5mC oxidation derivatives in different cell types of the developing and adult brain20
By revealing their spatial distribution20, this technique can be crucial to understanding the biological implications and the fate of oxidized derivatives of 5mC in various biological contexts where these modified derivatives of 5mC can be detected including cellular differentiation, development and disease. In addition, the method we describe here can give semi-quantitative assessments of the oxidized derivatives of 5mC in different tissues by assessing the kinetics of peroxidase reaction (which is proportional to the staining intensity) at different incubation times with tyramide26.

Methylation of cytosine bases in DNA (5mC) represents a major epigenetic mark found in vertebrates genomes associated with transcriptional silencing1. 5mC is being introduced and maintained by DNA methyltransferases2-5, and has been shown to play important roles in a number of biological processes including genomic imprinting, X-chromosome inactivation, cellular differentiation, and development3, 6. Consequently, the disruption of 5mC genomic patterns is associated with a number of diseases7, 8-11. Despite the progress in understanding 5mC role in development and disease, it is still remain largely unknown how this mark is being removed in developing and adult tissues. Several potential mechanisms of DNA demethylation have been recently proposed including active and passive demethylation mechanisms12, 13, 14, 15. Discovery of the products of 5mC sequential oxidation mediated by ten-eleven translocation enzymes (tet1/2/3) such as 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in eukaryotic DNA 16, 17, 18, 19 prompted speculations whether they may serve as intermediates of DNA demethylation processes or act as stable epigenetic marks in their own right13. While the demonstration that the component of base excision repair, thymine-DNA glycosylase (TDG) can bind and remove both 5fC and 5caC from DNA19, 20 suggests a role for the modified 5mC derivatives in an active DNA demethylation. Recent evidence showing that 5fC/5caC can modulate the rate of RNA II processivity points to potential involvement of these marks in transcriptional regulation29. Due to this potential biological importance of oxidized forms of 5mC, a range of biochemical and antibody based techniques have been employed for studying their genomic distribution and global content16, 19-24.
Given that most of the vertebrate organs consist of different cell types and that the distribution of modified cytosine bases is tissue and cell-type specific 16-18, 20, 23, 25-27, determining the spatial distribution of oxidized 5mC derivatives in different tissues becomes an important experimental task necessary for unveiling their biological functions. Most of the biochemical and antibody based approaches do not provide any spatial information regarding the distribution of the modified forms of 5mC in different tissue and cell-types. In contrast, immunochemistry based techniques can provide a rapid tool for assessing the spatial distribution and nuclear localization of 5mC28 and its oxidized derivatives20. That is said, the reported very low abundance of 5fC (20 in every 106 cytosine) and 5caC (3 in every 106 cytosine) in the mouse genome18 represents a significant challenge for standard immunochemistry.
Here, we describe a highly sensitive immunochemical method that provides robust and a rapid detection of oxidized form of cytosine in mammalian brain tissue. By incorporating peroxidase-conjugated secondary antibodies coupled with a signal amplification step, this method circumvents the challenges of detecting the very low amounts of 5fC and 5caC. In addition, this technique can be used to co-detect the modified forms of cytosine with lineage specific markers, effectively complementing other approaches in elucidating the biological functions of these epigenetic marks.

<table border="0" cellpadding="0" cellspacing="0" width="1281">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:388px;">Coplin jar</td>
			<td style="width:180px;">Cole-Parmer</td>
			<td style="width:119px;">UY-48585-30</td>
			<td style="width:595px;">Glass made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene</td>
			<td></td>
			<td></td>
			<td>Use only in Class II safety cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>12821680</td>
			<td>pPH 7.5, filtered before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% formaldehyde&#160; (FA)</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous denatured, histological grade</td>
			<td>Sigma Aldrich</td>
			<td>&#160;64-17-5&#160;&#160;</td>
			<td>95, 75, 50 % in PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.01 % Tween 20 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td>PBT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Triton X-100 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>PBX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 N HCl</td>
			<td>Sigma Aldrich</td>
			<td>71826</td>
			<td>caution extremely toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mM Tris-HCl&#160;</td>
			<td>Promega</td>
			<td>H5121</td>
			<td>PH adjusted to 8.5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrophobic barrier pen</td>
			<td>Abcam</td>
			<td>ab2601</td>
			<td>for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slide moisture chamber</td>
			<td>Scientific Device Laboratory</td>
			<td>197-BL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-5mC mouse antibody</td>
			<td>Diagenode</td>
			<td>C15200081</td>
			<td>monoclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5hmC antibody</td>
			<td>Active Motif</td>
			<td>39791</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5caC antibody</td>
			<td>Active Motif</td>
			<td>61225</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase-conjugated anti-rabbit seconday antibody</td>
			<td>Dako</td>
			<td>K1497</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">555-congjuated goat anti-mouse antibody</td>
			<td>Molecular probes</td>
			<td>A-11005</td>
			<td>&#160;secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% BSA</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td>Blocking solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22x32mm Glass cover slips</td>
			<td>BDH</td>
			<td>406/0188/24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyramide Signal Amplification System</td>
			<td>Perkin Elmer</td>
			<td>NEL741001KT</td>
			<td>Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colourless nail polish</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chicken polyclonal anti-GFAP polyclonal antibody</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">PA5-18598</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-NeuN mouse monoclonal antibody</td>
			<td>Merck milipore</td>
			<td style="width:119px;">MAB377B</td>
			<td>1:400 dilution</td>
		</tr>
	</tbody>
</table>

detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

To determine the distribution of 5hmC in brain tissue sections, we performed co-detection of this epigenetic modification with a marker for post-mitotic neurons, NeuN, employing commercial anti-5hmC antibody that specifically interacts with this mark but not with other forms of modified cytosine20, 25. Immunohistochemical analysis of 5hmC and 5caC distributions in the adult brain revealed that whereas the prominent 5hmC staining co-localizes with NeuN positive cells, NeuN-negative glial cells possess lower levels of genomic 5hmC (Figure 1)20.
We recently showed that Tet-dependent 5mC oxidation is operative during lineage specification of neural stem cells (NSCs) 20. Despite being immunochemically undetectable in NSCs, 5fC and 5caC exhibit prominent immunostaining at early stages of NSCs differentiation towards neuronal and glial lineages. Both these marks transiently accumulate concurrently with appearance of the markers of early neuronal and glial differentiation20. To determine the distribution of 5caC in differentiating NSCs we performed co-staining of this mark with a glial marker GFAP on the fixed cultures of NSCs at 3 days of induction of glial differentiation. Unlike in NSCs or mature astrocytes (data not shown), we observed strong 5caC signal in relatively large proportion of the cells expressing GFAP in these cultures (Figure 2)20.
<img alt="Figure 1" src="/files/ftp_upload/54416/54416fig1.jpg" /> 
Figure 1. Co-immunostaining of 5hmC (green) with NeuN (red) in the Adult Brain Tissue Counterstained with DAPI (Blue). Individual channels and the merged view are shown. Scale bars are 25 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54416/54416fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54416/54416fig2.jpg" /> 
Figure 2. Co-immunostaining of 5caC with GFAP in the Culture of NSC at Day 3 of Glial Differentiation. Individual channels and the merged view are shown. Scale bars are 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

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7.2K Views.  Université Libre de Bruxelles. Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Video: Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

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, ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.