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.