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.
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
In This Article
Summary
Abstract
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.
Introduction
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....
Access restricted. Please log in or start a trial to view this content.
Protocol
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
- Perfusion & fixation
- 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% paraformaldehy....
Access restricted. Please log in or start a trial to view this content.
Results
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.......
Access restricted. Please log in or start a trial to view this content.
Discussion
Standard IHC procedures often require specific adjustments to obtain an optimal result, which commonly implies extensive experience but also "trial and error" 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.......
Access restricted. Please log in or start a trial to view this content.
Disclosures
The authors declare no conflict of interest.
Acknowledgements
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.
....Access restricted. Please log in or start a trial to view this content.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Reagent | |||
| Isoflurane (Isofluran): 1-Chlor-2,2,2-trifluorethyl (difluormethyl ether) 2-Chlor-2-(difluormethoxy)-1,1,1-trifluorethan (C3H2ClF5O) | Baxter | 1001936060 | Eye irradiation. Probably influences fertility and damages baby in uterus. Administer only in adequately equipped anesthetizing environment. |
| Sodiumchloride (NaCl) | Merck | 1.0604 | |
| Phosphate Buffer Saline (PBS), tablet | Sigma-Aldrich | P4417 | |
| Paraformaldehyde (OH(CH2O)nH (n = 8 - 100)), 4% in 1x PBS | Apl pharma | 34 24 28 | Health hazard. Corrosive. Flamable. Acute toxicity. |
| Ethanol ≥99.5%, absolute (CH3CH2OH) | Sigma-Aldrich | 459844-1L | Flamable. |
| Xylene (Xylenes, histological grade; C6H4(CH3)2 | Sigma-Aldrich | 534056 | Flamable. Acute toxicity. |
| Histo-Comp Paraffin Wax | Tissue-Tek | V0-5-1001 | |
| Adhesive Microscope Slides | Starfrost | MIC-1040-W | |
| Xylol substitute XEM-200 | Vogel GmbH | ND-HS-200 | Respiratory sensitization. Carcinogenicity. |
| α-CD8 (Ox-8) mouse anti-rat, primary antibody | AbD Serotec | MCA48G | |
| α-Iba1 (AIF1) mouse anti-rat, primary antibody | Millipore | MABN92 | |
| α-CD68 (ED1) mouse anti-rat, primary antibody | AbD Serotec | MCA341R | |
| α-eotaxin C-19 (CCL11) goat anti-rat, primary antibody | Santa Cruz BT | SC-6181 | |
| Alkaline phosphatase (AP)-conjugated secondary antibody | Dakopatts, Denmark | D0314 | |
| Biotinylated secondary antibody | Amersham Biotech | RPN1025 | |
| Avidin- horseradish peroxidase complex (HRP) | Sigma-Aldrich | A3151 | |
| Naphthol AS-MX phosphate (C19H18NO5P) | Sigma-Aldrich | N4875-1G | Acute toxicity. |
| Fast Blue RR Salt, Azoic Diazo No. 24 (C15H14ClN3O3 x 1/2 ZnCl2) | Sigma-Aldrich | FBS25 | |
| Levamisol hydrochloride (C11H13ClN2S) | Sigma-Aldrich | 31742 | Acute toxicity. |
| 3,3'-Diaminobenzidine tetrahydrochloride (DAB Chromogen) | DAKO | S3000 | Highly flammable. Toxic. |
| Copper sulphate (CuSO4) | Merck | 1.02791 | Acute toxicity. Environmental hazzard. |
| GelTol Aqueous Mounting Medium | Thermo Electron Corporation | 230100 | |
| Hydrogen peroxide, 30% (H2O2) | Merck | 107210 | Acute toxicity. |
| Methanol (CH3OH) | Fluka | 65543 | Acute toxicity. Respiratory sensitization. Carcinogenicity. Flammable. |
| Tris (hydroxymethyl) aminomethane, TRIS base (C4H11NO3 ) | AppliChem | A1379 | Skin and eye irritation. |
| Tris Buffered Saline (TBS), tablet | Sigma-Aldrich | T5030 | |
| Di- Sodium hydrogen phosphate dihydrate (Na2HPO4 x 2H2O) | Merck | 1.0658 | |
| Sodium dihydrogen phosphate monohydrate (NaH2PO4 x 1H2O) | Merck | 1.06346 | |
| Fetal calf serum (FCS) | Cambrex BioScience | DE-14-802F | |
| DAKO cytomation wash buffer 10x | DAKO | S3006 | Should be stored at 2-8 °C to inhibit bacterial growth. Avoid foaming. |
| N,N-Dimethylformamide; DMF (C3H7NO) | Fluka | 40250 | Flammable. Acute toxicity. |
| Glas coverslips 24 x 36 mm | Menzel-Gläser | BB024036A1 | |
| Hydrochloric acid, conc. (HCl) | Sigma-Aldrich | 30721 | Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic. |
| Hydrochloric acid solution volumetric, 2 M HCl (2 N) | Fluka | 71826 | Corrosive, irritant, permeator. Lung sensitizer (as acid mist). Toxic. |
| Sodium nitrite, ReagentPlus, ≥99.0% (NaNO2) | Sigma-Aldrich | S2252 | Oxidant. Toxic. Dangerous for the environment. |
| Ethylenedinitrilotetraacetic acid disodium salt dihydrate (EDTA; C10H14N2Na2O8 x 2H2O) | Merck | 1.08454 | Oral exposures may cause reproductive and developmental effects. |
| Equipment | |||
| Tissue-TekV.I.P Vacuum Infiltration Processor | Sakura | 5902 VIP Jr. 115 V, 60 Hz | |
| Hacker-Bright 8000 Series Base Sledge Microtome | Hacker instruments | ||
| Household food steamer | Braun | MultiGourmet FS 20 | |
| Light microscope | Leica Polyvar 2 |
References
- Coons, A., Creech, H. J., Jones, R. N. Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med. 47, 200-202 (1941).
- Nakane, P. K., Pierce, G. B.
Access restricted. Please log in or start a trial to view this content.
Reprints and Permissions
