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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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

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 &#181;m 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 unspe...

Watch this Scientific Journal Video about Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections at JoVE.com

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

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 ...

immunohistochemical-analysis-rat-central-nervous-system-peripheral

Research

JoVE Journal

Neuroscience

17.0K Views.  Karolinska Institutet. 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.

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

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

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.

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...

Cancer Research

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

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

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.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

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

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

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.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Immunology and Infection

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

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

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.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

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.

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

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

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. 
To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated &#945;-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated &#945;-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.

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

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.

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

Biology

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

Source: Singh, P., et al. <a href="https://app.jove.com/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
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.

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
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