Name: Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections (Video) | JoVE Journal
Uploaded: 2016-11-14T12:30:00.000+00:00
Duration: 9 min 11 s
Description: Learn double immunostaining in rat central nervous system and peripheral lymph node tissue sections using formalin-fixed, paraffin-embedded samples. This protocol enables reliable protein localization visualization for neuroscience research.

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5 minutes

We thank Hans Lassmann and Jan Bauer for their guidance and support. We also thank Katalin Benedek for excellent technical assistance and Caroline Westerlund for critical and linguistic appraisal.
This study was supported by grants from Biogen Idec, the Wenner-Gren Foundation, the Swedish Research Council, the Swedish Association of Persons with Neurological Disabilities, Swedish Brain Foundation, the EU 6TH Framework EURATools (LSHG-CT-2005-019015) and Neuropromise (LSHM-CT-2005-018637). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Ethical Statement 
Present study is performed in accordance with guidelines from the Swedish National Board for Laboratory Animals and the European Community Council Directive (86/609/EEC) under the ethical permits N338/09, N15/10 and N65/10, which were approved by the North Stockholm Animal Ethics Committee.
1. Tissue Preparation
<ol>
	<li>Perfusion &#38; fixation
	<ol>
		<li>Anesthetize the animal with isoflurane to perform transcardial perfusion via left ventricle. Initiate the rinsing of vasculature with phosphate buffered saline (PBS) to remove the blood components, followed by 4% paraformaldehyde in 0.1 M PBS (PFA).</li>
		<li>Fixate dissected brains, spinal cords and peripheral LN tissue by immersing into PFA. After 24 hr at 4 &#176;C, transfer the tissue from PFA into PBS and store at 4 &#176;C until further processing. Alternatively to commercial PBS, dissolve 9 g NaCl in 250 ml the S&#246;rensen buffer and add 750 ml deionized water (dH2O); to prepare 0.2 M S&#246;rensen buffer (pH 7.4) dissolve 13.8 g NaH2PO4 x 1H2O and 71.2 g Na2HPO4 x 2H2O in 2.5 L dH2O.</li>
	</ol>
	</li>
	<li>Dehydration &#38; embedding
	<ol>
		<li>Cut the tissue into approximately 5 mm thick portions by using a razor blade.</li>
		<li>Initiate the tissue hardening process (dehydration) by using the Tissue-TekV.I.P Vacuum Infiltration Processor (standard procedure based on submersion into ascending concentrations of ethanol, followed by xylene and finally paraffin (Table 1).</li>
		<li>Place the tissue in a mold and pour in the liquid paraffin around the sample to form a &#34;paraffin block&#34;. Long-term storage requires room temperature (RT). However, prior to sectioning, it is recommendable to cool down the blocks, e.g., overnight (o/n) at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Sectioning
<ol>
	<li>Place the paraffin block into a fixed holder of the sledge microtome, which can move backwards and forwards across the knife. Adjust the optimal angle between the block and the microtome knife (that depends on knife geometry, but also on the cutting speed and technique).</li>
	<li>Cut 3 - 5 &#181;m thick cross-sections from the paraffin block.</li>
	<li>Transfer just cut section into a water container and mount it subsequently from the water onto the glass slide.</li>
	<li>Press the mounted section carefully against a paper towel to remove residual water and potential air bubbles. Commercially pre-coated adhesive glass slides are recommendable.</li>
	<li>Dry mounted slides for a couple of hours in a stove on 50 - 60 &#176;C.</li>
</ol>
3. Deparaffinization (Rehydration &#38; Blocking the Endogenous Peroxidase)
<ol>
	<li>Immerse the slides 2x into xylene (alternatively xylene substitute XEM-200), each time 15 - 20&#39;.</li>
	<li>Rinse in 99% ethanol.</li>
	<li>To block the endogenous peroxidase activity, incubate the sections for 30&#39; in methanol solution containing 0.25% hydrogen peroxide.</li>
	<li>Continue rehydration by using ethanol with increasing water content (99%, 70%, ending up in distilled water.</li>
	<li>From this point until mounting of the cover slips sections have to be kept moist.</li>
</ol>
4. Antigen Retrieval
<ol>
	<li>Use a food steamer for boiling the slides in an antigen retrieval solution (in this case EDTA pH 8.5 buffer) for 60&#39; (EDTA buffer stock solution contains 1.21 g Tris and 0.37 g EDTA dissolved in 50 ml dH2O; to prepare the working solution dilute 2.5ml EDTA stock solution in 47.5 ml dH2O).</li>
	<li>Cool down the slides on RT for approx. 1 hr and then rinse 3 - 5x with Tris buffered saline (TBS, use alternatively PBS) consisting of 0.05 M Tris and 0.15 M NaCl; pH 7.5 adjusted with HCl. Alternatively use commercial TBS.</li>
</ol>
5. Blocking the Unspecific Binding Sites
<ol>
	<li>To avoid unspecific background reactions, incubate the sections on RT for 30&#39; in the blocking solution containing 10% fetal calf serum (FCS) and 90% DAKO buffer.</li>
</ol>
6. Double Immunolabeling: Simultaneous Incubation with the Primary Abs
<ol>
	<li>Dilute required amount of primary antibodies in the blocking solution and incubate o/n at 4 &#176;C. For the double immunostaining combine &#945;-eotaxin (Ccl11; 1:300) with &#945;-Cd68 (Ed1; 1:1,000) or &#945;-Iba1 (Aif1, 1:1,000) or &#945;-Cd8&#945; (Ox-8; 1:200).</li>
	<li>Rinse the slides 3-5x with TBS buffer (use alternatively 10x diluted DAKO buffer).</li>
	<li>Dilute required amount of secondary antibodies (biotinylated anti-goat and AP-conjugated anti-mouse, both 1:200) in the blocking solution and incubate 1 hr on RT.</li>
	<li>Rinse the slides 3 - 5x with TBS buffer (use alternatively 10x diluted DAKO buffer).</li>
	<li>Incubate the slides with avidin-horseradish peroxidase complex (HRP) diluted in the blocking solution for 1 hr on RT.</li>
	<li>Rinse the slides 3 - 5x with TBS buffer (use alternatively 10x diluted DAKO buffer).</li>
</ol>
7. Visualization
<ol>
	<li>Visualization of the bound AP-labeled secondary antibody
	<ol>
		<li>Prepare 0.1 M Tris-HCl buffer by dissolving 12.1 g Tris in 1 L dH2O and adjust pH to by using HCl. Use the same Tris-HCl buffer to prepare 1 M levamisole solution. Prepare freshly 4% NaNO2 solution in dH2O.</li>
		<li>To obtain 50 ml of the Fast Blue (FB) substrate (volume required for one standard glass cuvette) dissolve 6.25 mg Naphtol-AS-MX-Phosphate in 312.5 &#181;l DMF in the glass tube and stir into 50 ml of pre-warmed (37 &#176;C) Tris-HCl buffer. To dissolve 12.5 mg FB RR Salt in 312.5 &#181;l 2 N HCl add 312.5 &#181;l of previously prepared 4% NaNO2 solution and stir the mixture into the same 50 ml of pre-warmed Tris-HCl buffer. Shake lightly until the yellow liquid becomes clear and finally add 77 &#181;l of previously prepared 1 M Levamisole solution. Filtrate obtained mixture and pour onto slides placed in the glass cuvette.</li>
		<li>Initiate the incubation at 37 &#176;C and control developing process under the light microscope approx. every 15 - 30 min. If turning fuzzy, replace the FB solution with the fresh mixture.</li>
		<li>Rinse the slides 3 - 5x with TBS buffer and transfer subsequently into PBS buffer.</li>
	</ol>
	</li>
	<li>Visualization of the bound biotinylated secondary antibody
	<ol>
		<li>Prepare DAB/H2O2 developing solution by diluting 1 ml DAB stock solution (25 mg DAB per 1 ml PBS) in 49 ml PBS. Add 16.5 &#181;l H2O2 and filtrate prior pouring onto sections.</li>
		<li>Conversion of the chromogen DAB into precipitating brown pigment can be immediate. Control the developing process under the light microscope (even couple of seconds longer incubation may raise a high background and mask the specific signal).</li>
		<li>The intensity of the brown pigment precipitate can be alternatively enhanced by incubation in the solution consisting of 2% copper sulphate and 0.9 % NaCl for 5&#39;.</li>
		<li>Rinse the slides 3 - 5x with PBS and finally with dH2O, use alternatively tap water.</li>
		<li>Mount the slides with cover slips directly from the water by using aqueous GelTol mounting medium. Avoid creating air bubbles.</li>
		<li>Allow complete drying of the mounting medium, e.g., o/n at 4 &#176;C. Store dried slides on RT.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.</td>
			<td>% Ethanol</td>
			<td>20&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>2.</td>
			<td>% Ethanol</td>
			<td>60&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>3.</td>
			<td>% Ethanol</td>
			<td>90&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>4.</td>
			<td>% Ethanol</td>
			<td>60&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>5.</td>
			<td>% Ethanol</td>
			<td>90&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>6.</td>
			<td>% Ethanol</td>
			<td>60&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>7.</td>
			<td>% Ethanol</td>
			<td>90&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>8.</td>
			<td>% Ethanol</td>
			<td>120&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>9.</td>
			<td>Xylol</td>
			<td>30&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>10.</td>
			<td>Xylol</td>
			<td>60&#39;</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>11.</td>
			<td>Paraffin</td>
			<td>60&#39;</td>
			<td>60 &#176;C</td>
		</tr>
		<tr>
			<td>12.</td>
			<td>Paraffin</td>
			<td>60&#39;</td>
			<td>60 &#176;C</td>
		</tr>
		<tr>
			<td>13.</td>
			<td>Paraffin</td>
			<td>60&#39;</td>
			<td>60 &#176;C</td>
		</tr>
		<tr>
			<td>14.</td>
			<td>Paraffin</td>
			<td>120&#39;</td>
			<td>60 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1. Tissue Processing by Tissue-TekV.I.P Vacuum Infiltration Processor.

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<li>Petrelli, F., Coderoni, S., Moretti, P., Paparelli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+biotin+on+phosphorylation%2C+acetylation%2C+methylation+of+rat+liver+histones%5BTitle%5D)+AND+%22Molecular+biology+reports%22%5BJournal%5D)">Effect of biotin on phosphorylation, acetylation, methylation of rat liver histones.</a> Molecular biology reports. 4, 87-92 (1978).</li>
<li>Yagi, T., Terada, N., Baba, T., Ohno, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+endogenous+biotin-containing+proteins+in+mouse+Bergmann+glial+cells%5BTitle%5D)+AND+%22The+Histochemical+journal%22%5BJournal%5D)">Localization of endogenous biotin-containing proteins in mouse Bergmann glial cells.</a> The Histochemical journal. 34, 567-572 (2002).</li>
<li>Van Hecke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Routine+Immunohistochemical+Staining+Today%3A+Choices+to+Make%2C+Challenges+to+Take%5BTitle%5D)+AND+%22Journal+of+Histotechnology%22%5BJournal%5D)">Routine Immunohistochemical Staining Today: Choices to Make, Challenges to Take.</a> Journal of Histotechnology. 1, 45-54 (2002).</li>
</ol>

The authors declare no conflict of interest.

Standard IHC procedures often require specific adjustments to obtain an optimal result, which commonly implies extensive experience but also &#34;trial and error&#34; approach. From tissue preparation until target visualization, almost each step in the protocol may be subjected to individually designed modifications in order to improve the final outcome. Double staining protocol presented here exemplifies IHC-based protein targeting particularly adjusted for assessing interrelations between the target proteins of our int...

Despite utilization of advanced high-throughput analyses performed on the methylome, transcriptome or even proteome level, immunostaining remains the golden standard for protein detection directly in the tissue sample, cell culture or a cell smear. By revealing the localization/distribution pattern, immunohistochemistry (IHC) may assess relative ratios and topographical interrelations of the target proteins, and even indicate their biological activities. Therefore, IHC is widely utilized for clinical and research purposes, e.g., for diagnosis, treatment evaluations, study of disease mechanisms, functional and phenotypical alterations in animal models, etc.
Essentially comprising histology, pathology, biochemistry and immunology, IHC has significantly advanced since 1941, when fluorescently labeled antibodies were used for the first time to identify Pneumococcal antigens in the infected tissue 1. Visualization of cellular products and components by IHC is based on binding of antibodies (Abs) to their specific antigen (Ag). Besides using fluorophore tagged antibodies, immune reactions can also be visualized by using enzymes like peroxidase 2,3 or alkaline phosphatase 4. Further, colloidal gold-tagged antibodies5 are used for detecting specific antigen-antibody interaction by both light and electron microscopy, while radioactive labels are visualized by autoradiography.
The Ag-Ab immunoreaction can be detected via direct and indirect methods. The direct method is essentially faster and simpler, as it uses directly labeled primary Abs 6. However, due to significant lack of sensitivity, indirect methods are preferred to the direct ones. Two-step indirect detection procedures require unlabeled primary Abs, as the first, and labeled secondary Abs directed against the primary Abs, as the second layer 7. Signal amplification can be achieved by involving further, enzyme-coupled tertiary Ab (three-step indirect method) that binds to the secondary Ab. Commonly used indirect detection methods are avidin-biotin and peroxidase-antiperoxidase (PAP). Alternatively, alkaline phosphatase-antialkaline phosphatase (APAAP) complex can be used instead of the PAP method. Notably, alkaline phosphatase (AP) methods appear to be even more sensitive than immunoperoxidase methods 4. Avidin-biotin complex (ABC) method uses biotinylated secondary Ab in combination with either labeled avidin-biotin complex (LAB), or labeled streptavidin-biotin complex (SLAB). Detection sensitivity can be further increased by involving avidin labeled with peroxidase or alkaline phosphatase 8. Other detection methods in use are polymeric labeling, tyramine amplification and immuno-rolling circle 9. Notably, different detection methods can be combined for multiple Ag detection in the same tissue sample, which was reported for the first time in 1978 4. Simultaneous double immunostaining presented here was performed in formalin-fixed, paraffin-embedded rat CNS and LN tissue sections using peroxidase-bound and AP-conjugated secondary antibodies, respectively. The signals were visualized using 3,3&#39;-diaminobencidine (DAB) chromogen and the Fast Blue (FB) APAAP complex, respectively.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Isoflurane (Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl ether) 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;ClF&lt;sub&gt;5&lt;/sub&gt;O)</td><td>Baxter</td><td>1001936060</td><td>Eye irradiation. Probably influences fertility and damages baby in uterus. Administer only in adequately equipped anesthetizing environment.</td></tr><tr><td>Sodiumchloride (NaCl)</td><td>Merck</td><td>1.0604</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS), tablet</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Paraformaldehyde (OH(CH&lt;sub&gt;2&lt;/sub&gt;O)&lt;sub&gt;n&lt;/sub&gt;H (n = 8 - 100)), 4% in 1x PBS</td><td>Apl pharma</td><td>34 24 28</td><td>Health hazard. Corrosive. Flamable. Acute toxicity.</td></tr><tr><td>Ethanol &amp;#8805;99.5%, absolute (CH&lt;sub&gt;3&lt;/sub&gt;CH&lt;sub&gt;2&lt;/sub&gt;OH)</td><td>Sigma-Aldrich</td><td>459844-1L</td><td>Flamable.</td></tr><tr><td>Xylene (Xylenes, histological grade; C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;(CH&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt; </td><td>Sigma-Aldrich</td><td>534056</td><td>Flamable. Acute toxicity.</td></tr><tr><td>Histo-Comp Paraffin Wax</td><td>Tissue-Tek</td><td>V0-5-1001</td><td></td></tr><tr><td>Adhesive Microscope Slides</td><td>Starfrost</td><td>MIC-1040-W</td><td></td></tr><tr><td>Xylol substitute XEM-200 </td><td>Vogel GmbH</td><td>ND-HS-200</td><td>Respiratory sensitization. Carcinogenicity.</td></tr><tr><td>&amp;alpha;-CD8 (Ox-8) mouse anti-rat, primary antibody</td><td>AbD Serotec</td><td>MCA48G</td><td></td></tr><tr><td>&amp;alpha;-Iba1 (AIF1) mouse anti-rat, primary antibody</td><td>Millipore</td><td>MABN92</td><td></td></tr><tr><td>&amp;alpha;-CD68 (ED1) mouse anti-rat, primary antibody</td><td>AbD Serotec</td><td>MCA341R</td><td></td></tr><tr><td>&amp;alpha;-eotaxin C-19 (CCL11) goat anti-rat, primary antibody</td><td>Santa Cruz BT</td><td>SC-6181</td><td></td></tr><tr><td>Alkaline phosphatase (AP)-conjugated secondary antibody</td><td>Dakopatts, Denmark</td><td>D0314</td><td></td></tr><tr><td>Biotinylated secondary antibody</td><td>Amersham Biotech</td><td>RPN1025</td><td></td></tr><tr><td>Avidin- horseradish peroxidase complex (HRP) </td><td>Sigma-Aldrich</td><td>A3151</td><td></td></tr><tr><td>Naphthol AS-MX phosphate (C&lt;sub&gt;19&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;NO&lt;sub&gt;5&lt;/sub&gt;P)</td><td>Sigma-Aldrich</td><td>N4875-1G</td><td>Acute toxicity.</td></tr><tr><td>Fast Blue RR Salt, Azoic Diazo No. 24 (C&lt;sub&gt;15&lt;/sub&gt;H&lt;sub&gt;14&lt;/sub&gt;ClN&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt; x 1/2 ZnCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>FBS25</td><td></td></tr><tr><td>Levamisol hydrochloride (C&lt;sub&gt;11&lt;/sub&gt;H&lt;sub&gt;13&lt;/sub&gt;ClN&lt;sub&gt;2S&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>31742</td><td>Acute toxicity.</td></tr><tr><td>3,3&#039;-Diaminobenzidine tetrahydrochloride (DAB Chromogen)</td><td>DAKO</td><td>S3000</td><td>Highly flammable. Toxic.</td></tr><tr><td>Copper sulphate (CuSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Merck</td><td>1.02791</td><td>Acute toxicity. Environmental hazzard.</td></tr><tr><td>GelTol Aqueous Mounting Medium</td><td>Thermo Electron Corporation</td><td>230100</td><td></td></tr><tr><td>Hydrogen peroxide, 30% (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck</td><td>107210</td><td>Acute toxicity.</td></tr><tr><td>Methanol (CH&lt;sub&gt;3&lt;/sub&gt;OH)</td><td>Fluka</td><td>65543</td><td>Acute toxicity. Respiratory sensitization. Carcinogenicity. Flammable.</td></tr><tr><td>Tris (hydroxymethyl) aminomethane, TRIS base (C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt; )</td><td>AppliChem</td><td>A1379</td><td>Skin and eye irritation.</td></tr><tr><td>Tris Buffered Saline (TBS), tablet</td><td>Sigma-Aldrich</td><td>T5030</td><td></td></tr><tr><td>Di- Sodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>1.0658</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; x 1H&lt;sub&gt;2&lt;/sub&gt;O) </td><td>Merck</td><td>1.06346</td><td></td></tr><tr><td>Fetal calf serum (FCS) </td><td>Cambrex BioScience</td><td>DE-14-802F</td><td></td></tr><tr><td>DAKO cytomation wash buffer 10x </td><td>DAKO</td><td>S3006</td><td>Should be stored at 2-8 &amp;deg;C to inhibit bacterial growth. Avoid foaming.</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide; DMF (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;7&lt;/sub&gt;NO)</td><td>Fluka</td><td>40250</td><td>Flammable. Acute toxicity.</td></tr><tr><td>Glas coverslips 24 x 36 mm </td><td>Menzel-Gl&amp;#228;ser</td><td>BB024036A1</td><td></td></tr><tr><td>Hydrochloric acid, conc. (HCl)</td><td>Sigma-Aldrich</td><td>30721</td><td>Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td></tr><tr><td>Hydrochloric acid solution volumetric, 2 M HCl (2 N)</td><td>Fluka</td><td>71826</td><td>Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td></tr><tr><td>Sodium nitrite, ReagentPlus, &amp;#8805;99.0% (NaNO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S2252</td><td>Oxidant. Toxic. Dangerous for the environment.</td></tr><tr><td>Ethylenedinitrilotetraacetic acid disodium salt dihydrate (EDTA; C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;14&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;Na&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>1.08454</td><td>Oral exposures may cause reproductive and developmental effects.</td></tr><tr><td colspan="4">&lt;strong&gt; Equipment&lt;/strong&gt;</td></tr><tr><td>Tissue-Tek&lt;sup&gt;V.I.P&lt;/sup&gt; Vacuum Infiltration Processor</td><td>Sakura</td><td>5902 VIP Jr. 115 V, 60 Hz</td><td></td></tr><tr><td>Hacker-Bright 8000 Series Base Sledge Microtome </td><td>Hacker instruments</td><td></td><td></td></tr><tr><td>Household food steamer </td><td>Braun</td><td>MultiGourmet FS 20</td><td></td></tr><tr><td>Light microscope </td><td>Leica Polyvar 2</td><td></td><td></td></tr></tbody></table>

immunohistochemical analysis in the rat central nervous system and peripheral lymph node tissue sections

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

Double immunostainings (co-stainings) were performed in formalin-fixed, paraffin-embedded rat CNS and LN sections. 3-5 µm thick tissue slices were cut using a sledge microtome, mounted subsequently onto pre-coated adhesive glass slides and treated as previously described 10,11,12. Briefly, after deparaffinizing, tissue rehydration and endogenous peroxidase inactivation, sections were subjected to the antigen retrieval process, followed by a blocking step to eliminate unspecific...

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

The overall goal of this procedure is to target a specific protein in the rat tissue. This is accomplished by first isolating, fixing, and paraffin-embedding the tissue, followed by sectioning of the tissue and mounting it onto slides. The second step is to deparaffinize and rehydrate the paraffin-embedded tissue sections, and then block the endogenous peroxidase.Next, the epitopes are uncovered by antigen retrieval. Subsequently, blocking is performed to reduce unspecific binding prior to introducing the antibodies. The final step is to detect the specific epitope by immunolabeling with primary and secondary antibodies, and to develop the signal from the labeled secondary antibody.Ultimately, the signal is visualized using light microscopy to show the protein localization pattern in the tissue sections. Though this method can provide insight into the protein localization and expression patterns in the rat central nervous system and in the peripheral lymph nodes, it can also be applied to other organs and species. For demonstrating the procedure will be a technician.Her name is Katalin Benedek, from the core facility of the microbiology tumor and cell biology center at the Karolinska Institutet. To begin tissue preparation, immerse the dissected brain, spinal cord, and peripheral lymph node tissue in four percent paraformaldehyde, and place it at four degrees Celsius for 24 hours. After the tissues are fixed, transfer the tissue into P-B-S and store it at four degrees Celsius until further processing.To begin processing the tissue, use a razor blade to cut the tissue into approximately five-millimeter sized portions. Then, initiate the tissue hardening process with a vacuum infiltration processor. The standard procedure is based on submersion into ascending concentrations of ethanol, followed by xylol, and finally paraffin.Next, place the tissue in a mold. Pour in the liquid paraffin around the sample to form a paraffin block, and store it at room temperature. Prior to sectioning the tissue, cool down the paraffin block overnight on the cold plate or in the refrigerator.After the paraffin block has been cooled, place the block into the fixed holder of a sledge microtome which can move backwards and forwards across a knife. Then, adjust the block and the microtome knife to the optimal angle, which depends on the knife geometry and the cutting speed and technique. Cut three five-micron thick cross-sections from the paraffin block.Then, transfer the cut sections into a water container and mount the sections from the water onto a commercial pre-coated adhesive glass slide. Next, press the mounted section carefully against a paper towel to remove residual water and potential air bubbles. Dry the mounted slides for a couple of hours in a stove set to 50 to 60 degrees Celsius.Once the slides are dry, immerse the slides two times in xylol for 15 to 20 minutes each. Then, rinse each slide in 99 percent ethanol. To block the endogenous peroxidase activity, incubate the sections for 30 minutes in a methanol solution containing 0.25 percent hydrogen peroxide.Then, continue rehydrating the tissue sections by rinsing with ethanol solutions containing increasing water content. Perform the final rehydration step in distilled water. Make sure to keep the sections moist until the cover slips are mounted.To retrieve the antigen, heat the slides with a household steamer and EDTA buffer at pH 8.5 for 60 minutes. Then, cool down the slides at room temperature for approximately one hour. Once the slides are cool, rinse each slide three to five times in Tris-buffered saline, or T-B-S.Next, incubate the tissue sections at room temperature for 30 minutes in a blocking solution containing 10 percent fetal calf serum and 90 percent wash buffer to block any non-specific background reactions. After the tissue sections have been blocked, dilute the required amount of primary antibodies in the blocking solution, and incubate the slides in this primary antibody solution overnight at four degrees Celsius. The next day, rinse the slides three to five times with T-B-S buffer.Then incubate the slides with secondary antibodies diluted in the blocking solution for one hour at room temperature. Afterwards, again rinse the slides three to five times with T-B-S buffer. Then, incubate the slides with avidin horseradish peroxidase complex, diluted one to 100 in the blocking solution, for one hour at room temperature.Once the one-hour incubation is complete, rinse the slides three to five times with T-B-S buffer. After preparing the fast blue solution as described in the text protocol, shake the dish lightly and filter the obtained mixture. Pour the solution onto the slides in the glass cuvette.Begin the incubation at 37 degrees Celsius, and observe the developing process under the light microscope every 15 to 30 minutes. Once the incubation is complete, rinse the slides three to five times with T-B-S buffer, and then subsequently transfer the slides into P-B-S buffer. To make the D-A-B hydrogen peroxide developing solution, first dilute one milliliter of a D-A-B stock solution in 49 milliliters of P-B-S.Add 16.5 microliters of hydrogen peroxide, and then filter the solution. Pour the solution onto the slides, and make sure to control the developing process under the light microscope in order to avoid high background that can mask the specific signal. Then, rinse the slides three to five times with P-B-S, followed by a final wash with distilled water or tap water.Mount the slides with cover slips directly from the water, using aqueous mounting medium. Avoid creating air bubbles between the cover slip and the slide. After mounting the sections, incubate the slides overnight at four degrees Celsius to allow the mounting medium to dry completely.Store the dried slides at room temperature. Double staining of peripheral lymph node tissue sections isolated from rats on day seven post-immunization revealed that cd8 positive t lymphocytes did not secrete the chemokine C-C-L 11. Alternatively, C-C-L 11 was found to co-localize with E-D one, a marker for lysosomal protein in activated macrophages.I-H-C double staining of a rat's spinal cord identified C-C-L eleven production in neuronal perikarya, dendrites, and axons, but not in I-B-A one positive macrophages and brain-resident microglia cells. In addition, the majority of E-D one positive macrophages and brain-resident microglia cells were also found to not be C-C-L 11 positive. After having watched this video, you should have a good understanding of how to perform a specific and reliable immunohistochemical targeting of the protein directly in tissue sections.

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Research

JoVE Journal

Neuroscience

This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Evilsizor, M. N., et. al., Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons. J. Vis. Exp. (2015)This video demonstrates a step-by-step immunohistochemistry staining procedure on cryosectioned rat brain tissue to visualize the spatial arrangement of microglia and neurons.

Immunohistochemical Staining of Rat Coronal Tissue Cryosections for Microglia and Neurons

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer’s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

Histological-Based Stainings Using Free-Floating Tissue Sections

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated α-synuclein is demonstrated in a mouse model of Parkinson's disease. This work also comprehensively describes the transcardial perfusion, dissection, tissue freezing/embedding, and frozen sectioning steps.

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Learn double immunostaining in rat central nervous system and peripheral lymph node tissue sections using formalin-fixed, paraffin-embedded samples. This protocol enables reliable protein localization visualization for neuroscience research.

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

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