Procedure for immunofluorescent staining of free-floating brain sections
TISSUE PREPARATION:
- Perfuse the anesthetized animal (rat or mouse) transcardially with ice-cold PBS (pH=7.4), followed by freshly made 4% paraformaldehyde fixative solution in PBS. If you have never done an animal perfusion enlist help from a local experienced individual and make sure that you follow applicable local care of animals guidelines.
- Remove brain and other tissues and postfix in the same 4% paraformaldehyde fixative solution in PBS at 4°C for 16 - 24 hours.
- Cryoprotect the tissue by immersing in sucrose solutions in PBS, first 15% sucrose, for 24 hours then 30% sucrose until tissue sinks, which may take from 48 hours up to 1 week. Sinking indicates that the tissue is fully impregnated with sucrose and so cryoprotected.
- Cut 40 - 50μm sections on a cryostat.
- Store sections in PBS + 0.05M NaN3 at 4°C until they were taken for staining. Sections should never be allowed to dry out during storage or staining.
IMMUNOFLUORESCENT STAINING:
- Rinse sections with PBS.
- Block and permeabilize sections in 10% normal goat serum or serum of the species the secondary antibodies were made in, in PBS plus 1% Triton-100 (PBSt) for 1 hour with slight agitation.
- Incubate sections with the primary antibody diluted in 1% normal goat serum in PBSt at 4°C overnight with slight agitation. Typical starting antibody concentration for pure antibody is 1mg/mL, though many will work well at much lower concentrations.
- Rinse sections 3 times with PBS, first rinse is quick, but wait 5 minutes between each subsequent rinses. This step removes unbound primary antibody.
- Add fluorochrome-conjugated secondary antibody, diluted 1:2,000, 0.5mg/mL, in 1% normal goat serum in PBSt. Can add Hoechst 33258 10mg/mL solution diluted 1:2,000, a convenient fluorescent blue dye for nuclear DNA staining. Cover specimen with foil and incubate for 2 hours at room temperature with slight agitation. We use ALEXA or Dylight dyes. The ALEXA dyes are sulphonated rhodamine compounds and are more stable to UV than FITC, TRITC, Texas red etc. while Dylight dyes are claimed be generally superior to ALEXA dyes. Both are marketed by ThermoFisher. We typically incubate for 1hr at 37°C or 2 hours at room temperature or overnight at 4°C.
- Rinse sections 4 times with PBS, first rinse is quick, but wait 5-10 minutes between each subsequent rinse.
- Mount sections on clean glass slides with glycerol-base mounting medium, and cover with coverslip. Southern Biotech, Vector labs and other vendors supply fluorescent sample mounting media which contain DAPI, a close relative of Hoechst 33258 mentioned in step 5 above. DAPI and Hoechst 33258 are both strong fluorescent DNA stains so if you use mounting media with DAPI you don’t need to add Hoechst in step 5 and vice versa. Store slides at 4°C, view on a suitable fluorescent, confocal or multiphoton microscope.