Immunostaining with fluorescent secondary antibodies for frozen sections
1. Prepare frozen sections from your tissue
2. Mount it on L-PolyLysine Slides.
3. Wash sections for 30 seconds in cold Acetone (-20°C). This step allows better antibody penetration (may wash out your antigen, especially in the case of cytosolic proteins). Let section dry before next step (in hood).
3. block non-specific binding by incubation of section with 1%-0.5% goat serum in PBS for 1 hour at 37°C.
4. Draw circle round dry section with PAP pen to prevent loss of antibody. Add primary antibodies; Typically dilute primaries to 1mg/ml and make up in PBS plus 0.1% goat serum. Usualy for double staining you can apply chicken, rabbit and mouse antibodies at same time. I prefer to do it setep by step.Typically incubate for 1 hour at 37°C or 2 hours at room temperature or overnight at 4°C.
5. Wash sections at least 3 times, at least 5 minutes each time, in PBS. To reduce background can include 0.1% Tween 20 in PBS.
6. Apply secondary antibodies. Best are goat anti-mouse and goat anti-rabbit antibodies from Molecular Probes, or Jackson. Typically incubate for 1hr at 37°C or 2 hours at room temperature at a dilution 1:5000.
7. Wash sections at least 3 times, at least 5 minutes each time, in PBS.
8. Mount in mounting medium, a useful one being Vectasheild mounting medium with DAPI, which allows you to stain nuclei with the DNA intercalating fluorescent dye DAPI, which you can see on a fluorescence microscope fitted with appropriate blue filters.
9. View on fluorescence microscope.