TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina (4) Confocal microscopy
Lab/Group: Egwuagu Lab (NIH)
** An editorial error was made: Cheng-Rong Yu prepared this manuscript and should have been the first author.
Related Journal & Article Information
Journal: Nature Medicine
Article Title: TH17 cells are expanded by IL-2 during Uveitis or Scleritis and inhibited by IL-27/STAT1 pathways
Introduction
This protocol is for histology and immunohistochemistry.
Other protocols related to our Nature Medicine paper can be found here:
Detection of TH17 cells in human blood
Analysis of the expression of IL-17 in mouse PBMC, lymph node and retina
Materials
Reagents
• Immunolabeling buffer (1X PBS, 0.5% BSA, 0.2% Tween-20, 0.05% NaN3, pH 7.3).
• Alexa 488 conjugated goat anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA)
• DAPI (1ug/ml) (Invitrogen).
• Rabbit polyclonal IL-27p28 antibody (IMG-5096A-Imgenex, San Diego, CA)
• Normal goat serum
• Vectashield mounting medium
• Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA)
• on SuperFrost/ Plus slides (Fisher Scientific, Pittsburgh, PA)
• Anti-fading agent (Gel/Mount, BioMeda, Foster City, CA)
Equipment
• Leica SP2 laser scanning confocal microscope (Leica Microsystems, Exton, PA)
• Leica 3060S cryostat
Procedure
Histology and immunohistochemical analysis
1| Euthanize adult mice (C57bl/6 strain) by CO2 and embed globes in cryomolds containing Tissue-Tek OCT compound and frozen on dry ice.
2| Cut frozen sections (10 µm thickness) with a Leica 3060S cryostat and collect on SuperFrost/ Plus slides and store at -70°C.
3| Prior to immunolabeling, allow slides to air dry under vacuum for 20 minutes.
4| Fix sections for 10 minutes in 4% formaldehyde in 1X PBS, wash repeatedly in 1X PBS, prior to blocking in 5% normal goat serum in immunolabeling buffer
5| For paraffin embedded sections fix eyes in 10% Formalin (overnight)
6| Embed globe in paraffin and cut 5μm sections with a microtome
7| Paraffinize sections used for antibody staining as follows:
- Incubate in xylene for 3 minutes each (3X).
- Incubate in 100% ethanol for 2 minutes (2X).
- Hydrate by placing slide in 95% ethanol for 2 minutes (2X)
- Then 70% ethanol for 2 minutes (2X)
- Then 50% ethanol for 2 minutes (2X)
- Then 30% ethanol for 2 minutes (2X)
- Transfer sections to distilled water.
8| Block with normal rabbit serum for 1 hr.
9| Dilute rabbit polyclonal antibody to IL-27 to 5 and 10µg/ml in immunolabeling buffer (stock antibody =500ug/ml/ working dilutions of 1:50 and 1:100).
10| Incubate sections in diluted primary antibody overnight at 4°C in a humidified chamber.
11| Rinse sections in immunolabeling buffer (3X)
12| Then incubate in Alexa 568 conjugated goat anti-rabbit secondary antibody containing DAPI (1µg/ml) for 1hr at room temperature in an opaque chamber.
13| Wash sections in immunolabeling buffer (3X)
14| Then mount in anti-fading agent and cover with cover-slip. Primary antibody was omitted from sections used as negative controls.
15| Examine sections on a Leica SP2 laser scanning confocal microscope
16| All images were collected under identical PMT detector settings.
Troubleshooting
Critical Steps
Anticipated Results
References
1. Nussenblatt, R.B. Proctor Lecture. Experimental autoimmune uveitis: mechanisms of disease and clinical therapeutic indications. Invest Ophthalmol Vis Sci 32, 3131-3141 (1991).
2. Gregerson, D.S., Obritsch, W.F., Fling, S.P. & Cameron, J.D. S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis. Journal of immunology (Baltimore, Md.) 136, 2875-2882 (1986).
3. Wacker, W.B., Donoso, L.A., Kalsow, C.M., Yankeelov, J.A., Jr. & Organisciak, D.T. Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina. Journal of immunology (Baltimore, Md.) 119, 1949-1958 (1977).
4. Egwuagu, C.E., Mahdi, R.M., Nussenblatt, R.B., Gery, I. & Caspi, R.R. Evidence for selective accumulation of V beta 8+ T lymphocytes in experimental autoimmune uveoretinitis induced with two different retinal antigens. J Immunol 151, 1627-1636. (1993).
5. Pepperberg, D.R., Okajima, T.L., Ripps, H., Chader, G.J. & Wiggert, B. Functional properties of interphotoreceptor retinoid-binding protein. Photochem Photobiol 54, 1057-1060 (1991).
6. Takase, H., et al. Induction of suppressors of cytokine signaling (SOCS) in the retina during experimental autoimmune uveitis (EAU): potential neuroprotective role of SOCS proteins. J Neuroimmunol 168, 118-127 (2005).
7. Yu, C.R., et al. Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells. J Biol Chem 278, 29752-29759 (2003).
8. Egwuagu, C.E., et al. Suppressors of cytokine signaling proteins are differentially expressed in Th1 and Th2 cells: implications for Th cell lineage commitment and maintenance. J Immunol 168, 3181-3187. (2002).
Acknowledgements
This research was funded by the Intramural Research Programs of the NEI and NIH. Authors thank Dr. Wei Zhu (Laboratory of Immunology, NEI, NIH) for assistance with fundoscopy.
Keywords
experimental autoimmune uveoretinitis (EAU); IL-27p28 immunohistochemistry and confocal microscopy; Intracellular cytokine staining assay; Retinal organ culture