Free floating immunofluorescent staining protocol on mouse brain sections

Briefly, the mouse brain tissue sections are prepared by washing off the cryoprotectant medium and then antigen retrieval is performed followed by quenching, blocking and primary antibody incubation. Sections are then washed and incubated in the appropriate secondary antibody solution and are then mounted, cover-slipped and sealed.

30 um mouse brain sections were stored in anti-freeze solution at -20°C until required.

1. Remove samples from freezer and equilibrate at Room temperature for 0h 10m 0s - 0h 20m 0s
2. Pour sections into a well insert in a 6-well plate to separate storage solution from section
3. Move the well insert to another well containing approximately 6mL of 1x PBS. Wash at least 5x with 1x PBS for 0h 5m 0s each on an orbital shaker using low speed at Room temperature

Incubate the sections in 10mM sodium citrate buffer (pH 6.0) for 0h 30m 0s. Let it cool to Room temperature1. Rinse the sections 3x0h 5m 0s each in 1X PBS

Weigh NaBH₄to make 0.1~0.5% in 1X PBS, made fresh1. Move the insert with sections into the fresh-made solution for 0h 30m 0s at Room temperature

1. Wash 2x 0h 5m 0s in 1X PBS

Incubate sections in normal donkey serum IF blocking buffer2h 0m 0s Room temperature on shaker 60 rpm

Make primary antibody cocktails in blocking buffer

1. Prepare ~300µL per sample of primary antibody solution consisting of selected primary antibody (diluted appropriately) in home-made normal donkey serum IF blocking buffer
2. Transfer sections from well insert into wells of black porcelain spot plate containing primary antibody solution to bind to the antigen(s) of interest
3. Place the plate on a rotating mixer using low speed (speed 7 rpm) and incubate 72h 0m 0s at 4°C (or 3X night/ over weekend)

The following day, pour sections into a well insert in a 6-well plate to separate sections from primary antibody solution.1. Wash sections 3 times with 1x PBST at Room temperature. Note: 0h 0m 30s for the first two rinses, 3x 0h 10m 0s for additional washing

1. Prepare 300µL per sample of secondary antibody solution consisting of appropriate secondary antibody + Hoechst 33342 (diluted accordingly) in blocking buffer (shield solution from light)
2. Transfer sections into the black porcelain spot plate containing 300µLsecondary antibody cocktail
3. Incubate for 2h 0m 0s at Room temperature on orbital shaker using low speed (shield solution from light).
4. Pour sections into a well-insert in a 6-well plate containing 1X PBST to separate sections from the secondary antibody solution
5. Continuing to shield samples from light, wash 3 times with 1x PBS for 0h 5m 0s at Room temperature

Pour sections into a glass petri dish1. Submerge a glass slide into the 1x PBS and use a fine paintbrush to coax the sections towards the slide

1. Gently tap the sections onto the slide, making sure there are no wrinkles or folds
2. Repeat until all sections are mounted onto the slide(s)

After sections are dried onto the slide(s), about 0h 15m 0s at Room temperature or until sections look opaque (remember to shield slides from light), apply an appropriate aqueous mounting medium (hardening or non-hardening). Antifading (DAKO Fluorescence Mounting Medium is preferred if using a fluorescent conjugated secondary antibody1. Using tweezers, place a coverslip on top of the medium. Cover with filter paper and press down firmly to remove excess mounting medium

1. Image sections using an appropriate microscope. Store in a dark slide box at 4°C