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 border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Isoflurane 			(Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl 			ether) 
 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan 			(C3H2ClF5O)</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(CH2O)nH (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 			&ge;99.5%, absolute (CH3CH2OH)</td>
 <td>Sigma-Aldrich</td>
 <td>459844-1L</td>
 <td>Flamable.</td>
 </tr>
 <tr>
 <td>Xylene 			(Xylenes, histological grade; C6H4(CH3)2 </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>&alpha;- CD8 			(Ox-8) mouse anti-rat, primary antibody</td>
 <td>AbD 			Serotec</td>
 <td>MCA48G</td>
 <td></td>
 </tr>
 <tr>
 <td>&alpha;- Iba1 			(AIF1) mouse anti-rat, primary antibody</td>
 <td>Millipore</td>
 <td>MABN92</td>
 <td></td>
 </tr>
 <tr>
 <td>&alpha;- CD68 			(ED1) mouse anti-rat, primary antibody</td>
 <td>AbD 			Serotec </td>
 <td>MCA341R</td>
 <td></td>
 </tr>
 <tr>
 <td>&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 (C19H18NO5P)</td>
 <td>Sigma-Aldrich</td>
 <td>N4875-1G</td>
 <td>Acute 			toxicity.</td>
 </tr>
 <tr>
 <td>Fast Blue 			RR Salt, Azoic Diazo No. 24 (C15H14ClN3O3 			x 			1/2 ZnCl2)</td>
 <td>Sigma-Aldrich</td>
 <td>FBS25</td>
 <td></td>
 </tr>
 <tr>
 <td>Levamisol 			hydrochloride (C11H13ClN2S)</td>
 <td>Sigma-Aldrich</td>
 <td>31742</td>
 <td>Acute 			toxicity.</td>
 </tr>
 <tr>
 <td>3,3'-Diaminobenzidine 			tetrahydrochloride (DAB Chromogen)</td>
 <td>DAKO</td>
 <td>S3000 </td>
 <td>Highly 			flammable. Toxic.</td>
 </tr>
 <tr>
 <td>Copper 			sulphate (CuSO4)</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% (H2O2)</td>
 <td>Merck</td>
 <td>107210</td>
 <td>Acute 			toxicity.</td>
 </tr>
 <tr>
 <td>Methanol 			(CH3OH)</td>
 <td>Fluka</td>
 <td>65543</td>
 <td>Acute 			toxicity. Respiratory sensitization. Carcinogenicity. Flammable.</td>
 </tr>
 <tr>
 <td>Tris 			(hydroxymethyl) aminomethane, TRIS base (C4H11NO3 			)</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 (Na2HPO4 			x 			2H2O)</td>
 <td>Merck</td>
 <td>1.0658</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium 			dihydrogen phosphate monohydrate (NaH2PO4 			x 			1H2O) </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 &deg;C to inhibit bacterial growth. Avoid foaming.</td>
 </tr>
 <tr>
 <td>N,N- 			Dimethylformamide; DMF (C3H7NO)</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&auml;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, 2M HCl (2N) </td>
 <td>Fluka </td>
 <td>71826</td>
 <td>Corrosive, 			irritant, permeator. Lung sensitizer (as acid mist). Toxic.</td>
 </tr>
 <tr>
 <td>Sodium 			nitrite, ReagentPlus, &ge;99.0% (NaNO2)</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; C10H14N2Na2O8 			x 2H2O)</td>
 <td>Merck</td>
 <td>1.08454</td>
 <td>Oral 			exposures may cause reproductive and developmental effects.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td> Equipment</td>
 </tr>
 <tr>
 <td>Tissue-Tek 			V.I.P 			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

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

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

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In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY