an immunohistochemistry staining for the detection of rabies virus antigens

An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens

Using a microtome, make a 5-micrometer paraffin section. Float the section on a water bath at 38 degrees Celsius, and collect it onto glass slides. Label the slides with a reagent-resistant pen. Place the slides onto a tray, and melt in an oven at 55 to 60 degrees Celsius for one hour.
Then, remove the slides from the oven, and immediately deparaffinize them using three consecutive xylene rinses of five minutes each. After this, rehydrate the sections on the slide by sequential immersions in decreasing dilutions of ethanol to deionized water, as outlined in the text protocol.
Treat the slides with the protease for 30 minutes for proteolytic antigen retrieval, then, rinse the slides in PBST for 10 minutes, and treat them with 3% hydrogen peroxide for 10 minutes. After this, wash the slides again with PBST for 10 minutes.
To begin, remove one slide from the buffer, making sure to only handle one slide at a time, while the others remain submerged, and use a paper towel to blot off excess buffer from around the tissue section. Place the slides into a humidity chamber, and incubate with normal goat serum at room temperature for 15 minutes.
Next, incubate the slides in the humidity chamber with the optimal predetermined dilution primary anti-rabies antibody and negative control antibodies at room temperature for 60 minutes with no washes in between. After this, wash the slides with PBST for 10 minutes, and incubate in a humidity chamber with the biotinylated antibody at room temperature for 15 minutes.
Wash the slides again with PBST for 10 minutes. Incubate the slides in a humidity chamber with streptavidin-HRP complex at room temperature for 15 minutes, and wash the PBST for 10 minutes. Then, incubate with AEC in a humidity chamber at room temperature for 10 minutes. Wash the slides in deionized water for 10 minutes and counterstain with Gill&#39;s hematoxylin diluted 1 to 2 with deionized water for two minutes.
After this, rinse off the excess hematoxylin by dip-rinsing the slides in deionized water. Rinse the slides in Scott&#39;s tap water for 30 seconds, and wash in deionized water for 10 minutes. Remove the slides one at a time and mount them with water-soluble mounting medium. Then, use a light microscope to read the slides.

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Nanyang Technological University

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Formalin fixation of tissues
<ol>
	<li>Place 3-5 mm brain tissues collected after necropsy&#160;into 10% phosphate-buffered formalin solution (1:20 to 1:50 tissue/formalin ratio) for 24-72 h. 
	CAUTION: Formalin is a toxic fixative.</li>
	<li>Record the approximate tissue weight (size), tissue type (brain areas), and volume of formalin. It is important for laboratories to document and maintain records on fixation times.</li>
	<li>For longer tissue storage after formalin fixation and prior to processing, place the tissue in 70% ethanol.</li>
</ol>
2. Tissue processing
<ol>
	<li>Following the specimen fixation, dissect the tissue to include the important brain areas e.g., cross sections of the brainstem, cerebellum (3 lobes), or the hippocampus (both hippocampi) each cut 3 to 5 mm thick and placed into processing cassettes.</li>
	<li>Process the tissue cassettes for paraffin wax infiltration, embedded into paraffin blocks and sectioned (3 to 6 &#181;m) on a microtome.</li>
</ol>
3. Preparation of Materials / Staining dishes
<ol>
	<li>Set up the staining dish (Table of Materials) as shown in&#160;Figure 1. Fill each dish with 250 mL of the solution.</li>
	<li>Preparation of the 3-Amino-9-ethylcarbizole (AEC) substrate stock solution
	<ol>
		<li>Dissolve one 20 mg tablet of 3-amino 9-ethylcarbazole (AEC) into 5 mL of N,N, dimethylformamide using a glass pipette.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		CAUTION: AEC is a carcinogen.</li>
	</ol>
	</li>
	<li>Preparation of the protease (e.g., Pronase) stock solution for the antigen retrieval
	<ol>
		<li>Dissolve 7 mg of the protease in 200 mL of phosphate-buffered saline (PBS).</li>
	</ol>
	</li>
	<li>Preparation of the rinse buffer PBS-T
	<ol>
		<li>Add 10 mL of Tween 80 to 990 mL of PBS. Mix it well to form a homogenous solution.</li>
	</ol>
	</li>
</ol>
4. Deparaffinization and tissue rehydration
<ol>
	<li>Make a 5 &#181;m paraffin section using a microtome, float it on a water bath at 38 &#730;C, and collect it onto glass slides. Label the slides with a reagent-resistant pen/marker.</li>
	<li>Place the slides onto a tray and melt in a 55-60 &#730;C oven for 1 hour. Do not raise the temperature above 60 &#730;C as it can destroy the viral antigen.</li>
	<li>Remove slides from the oven and immediately deparaffinize in 3 consecutive xylene rinses of 5 min each in dishes 1, 2, and 3.</li>
	<li>Rehydrate the sections on the slide by sequential immersions in decreasing dilution of ethanol to deionized water: (4 through 11 is a dip rinse) dish 4: xylene/100% ethanol (1:1); dish 5: 100% ethanol; dish 6: 100% ethanol; dish 7: 95% ethanol; dish 8: 95% ethanol; dish 9: 80% ethanol; dish 10: 70% ethanol; dish 11: deionized water (Figure 1. Dish set-up). 
	CAUTION: Xylene is a hazardous chemical and work should be conducted in a fume hood.</li>
</ol>
5. Proteolytic antigen retrieval
<ol>
	<li>Treat slides with the protease (2.5 &#181;g/mL of PBS) for 30 min for proteolytic antigen retrieval in dish 12.</li>
	<li>Then rinse in PBS-T for 10 min (dish 13).</li>
	<li>Treat with 3% hydrogen peroxide for 10 min (dish 14).</li>
	<li>Again, wash with PBS-T for 10 min (dish 15).</li>
</ol>
6. Staining procedure
<ol>
	<li>Handle slides one at a time keeping the remaining slides submerged in the buffer (do not remove the whole slide holder - keep slides wet). Remove one slide and blot off excess buffer (using a paper towel) from around the tissue section being careful not to disturb the tissue section. Incubate slides in a humidity chamber, made by placing moistened paper towels, on the lab bench top&#160;at room temperature with normal goat serum (blocking) for 15 min.</li>
	<li>Incubate with the optimal pre-determined dilution primary anti-rabies antibody (1:250 dilution of mouse anti-rabies serum, unpublished) (positive control) and negative control antibodies at room temperature same as above (step 6.1.) for 60 min with no washes in between.</li>
	<li>After 60 min, wash with PBS-T for 10 min (dish 16).</li>
	<li>Incubate with the biotinylated antibody (species-specific) in a humidity chamber at room temperature for 15 min (handling same as step 6.1.).</li>
	<li>Wash with PBS-T for 10 min (dish 16).</li>
	<li>Incubate with Streptavidin- horseradish peroxidase (HRP) complex in a humidity chamber at room temperature for 15 min (handling same as 6.1.).</li>
	<li>Wash with PBS-T for 10 min (dish 16).</li>
	<li>Incubate with peroxidase substrate, amino-ethylcarbazole (AEC), in a humidity chamber at room temperature for 10 min. Make AEC just prior to use. To do so, add 1 mL of AEC stock solution to 14 mL of 0.1M acetate buffer, pH 5.2. Add 0.15 mL of 3% peroxide, H2O2. Filter the mixture just before use (0.45 &#181;m filter). 
	NOTE: The working solution of AEC is only stable for 2-3 hours. The AEC stock solution can be stored in the refrigerator for longer periods.</li>
	<li>Wash in deionized water for 10 min (dish 17)</li>
	<li>Counterstained with Gill&#39;s Hematoxylin diluted 1:2 with deionized water for 2 min (dish 18).</li>
	<li>Rinse off excess Hematoxylin with deionized water dip rinse (dishes 19 and 20).</li>
	<li>Rinse in Scott&#39;s Tap water for 30 s (bluing solution) dish 21.</li>
	<li>Wash in deionized water for 10 min (dish 22)</li>
	<li>Remove slides one at a time - mount with a water-soluble mounting medium.</li>
	<li>Read slides on a light microscope.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% hydrogen peroxide</td>
			<td>Pharamacy brands</td>
			<td></td>
			<td>Off the shelf 3% H2O2</td>
		</tr>
		<tr>
			<td>3-Amino-9-ethylcarbazole (AEC)</td>
			<td>Millipore Sigma</td>
			<td>A6926</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate Buffer pH 5.2</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s140</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffered Formalin 10% Phosphate Buffered</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td>Certified</td>
		</tr>
		<tr>
			<td>Cover slips Corning</td>
			<td>Fisher Scientific</td>
			<td>12-553-471</td>
			<td>24 X 50 mm</td>
		</tr>
		<tr>
			<td>Ethanol 190 Proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000190</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill&#39;s hematoxylin formulation #2</td>
			<td>Fisher Scientific</td>
			<td>CS401-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoMark Biotin-Streptavidin Peroxidase Kit</td>
			<td>seracare</td>
			<td>71-00-18</td>
			<td>Mouse Primary Antibody&#160;</td>
		</tr>
		<tr>
			<td>ImmunoHistoMount</td>
			<td>Millipore Sigma</td>
			<td>i1161</td>
			<td>Mounting media</td>
		</tr>
		<tr>
			<td>N,N, Dimethyl formamide GR</td>
			<td>Fisher Scientific</td>
			<td>D119</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline&#160;</td>
			<td>HyClone</td>
			<td>RR14440.01</td>
			<td>01M, pH 7.2 (pH 7.2-7.6)</td>
		</tr>
		<tr>
			<td>Plan-APOCHROMAT 40X/0.95 Objective</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plan-APOCHROMATIC 20X/0.75 Objective</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Millipore Sigma</td>
			<td>53702</td>
			<td>Protease,&#160;Streptomyces griseus</td>
		</tr>
		<tr>
			<td>Scott&#39;s Tap Water&#160;</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Slide stain set</td>
			<td>Fisher Scientific</td>
			<td>50-294-72</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN-80&#160;</td>
			<td>Millipore Sigma</td>
			<td>P1754</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X3S-4</td>
			<td>Histological Grade</td>
		</tr>
		<tr>
			<td>Zeiss Axioplan 2 imaging - microscope</td>
			<td>Multiple vendors</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Niezgoda, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60138">Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues.</a> J. Vis. Exp. (2021)
This video demonstrates immunohistochemistry staining for rabies virus antigen detection in infected mouse brain tissue. The process includes deparaffinization and alcohol treatment, followed by proteolytic treatment to enhance antigen accessibility. Immunohistochemistry staining reveals red precipitates, confirming the presence of rabies antigens in the tissue section.

<img alt="Figure 1" src="/files/ftp_upload/21982/21982_Figure1.jpg" /> 
Figure 1: Flow chart indicating different steps for IHC testing.&#160;

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Formalin fixation of tissues

	...

Begin with a slide carrying a formalin-fixed, paraffin-embedded rabies virus-infected mouse brain tissue section. The fixed section retains viral antigens via protein cross-linking.
Treat the slide with a xylene solution to remove the paraffin.
Immerse the slide in decreasing ethanol concentrations to rehydrate the tissue, making it compatible with aqueous solutions.
Introduce a protease-containing buffer to disrupt protein cross-links, improving antigen accessibility.
Submerge in hydrogen peroxide to block endogenous peroxidases, and apply a blocking solution to prevent non-specific interactions.
Introduce primary antibodies specific to the rabies antigen, forming antigen-antibody complexes.
Add biotinylated secondary antibodies, which bind to the primary antibodies. Incubate with streptavidin-conjugated peroxidase enzymes, which interact with biotin.
Introduce a peroxidase substrate, producing a magenta-red precipitate.
Counterstain the tissue with a nuclear&#160; stain, followed by a bluing agent, staining the nuclei&#160; blue.
The immunohistochemistry image reveals magenta-red spots in the cytoplasm, confirming the presence of rabies antigens in the tissue section.

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An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens

Transkrypcja

An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens