Immunohistochemistry protocol for cryostat sections Inma Cobos 7/04
Immunohistochemistry protocol for cryostat sections
Inma Cobos 7/04
Fixation and cryoprotection:
1 Fix embryos overnight at 4 C in 4% paraformaldehyde, 0.1M PB.
2 Transfer embryos to 30% sucrose in 0.1M PB for several hours (after it sinks),
3 Transfer the embryos to well plates, suck the sucrose off and add OCT. Stir. Put the plate on the shaker at 4
C for 1 hour (stir them with a plastic pipette a few times to help the OCT embed the tissue). Transfer the
embryos to plastic molds and freeze the blocks on dry ice. Blocks can be stored at -80 C several months if
necessary.
4 Cut 20 ¦Ìm sections on the cryostat and mount the sections on fisherbrand superfrost plus slides (No 12550-15).
6 After sectioning put the slides into a clean slide box with some desiccant caps. Put the box in a plastic zip
bag at ¨C80 C.
Intracardiac perfusion with 4% Paraformaldehyde (PFA) in PBS and postfixation in the same fixative for 3
hrs
Cryoprotection with 30% sucrose for 1 day (after the brains sink).
Sectioning
4 Cut 10-20 ¦Ìm sections on the cryostat and mount the sections on fisherbrand superfrost plus slides (No 12550-15).
6 After sectioning the slides can be put into a slide box with some desiccant caps and stored (in a plastic zip
bag) at ¨C80 C.
DAY 1
Immunohistochemistry
3x5' PBS
1x10' 0.2% Triton X-100 (Sigma) in PBS (PBS-Tx 0.2%)
Endogenous Perioxidase Blockade (PROTECT FROM THE LIGHT WITH ALUMINIUN FOIL)
1x20' incubation in:
- 0.5% Hydrogen peroxide in PBS-Tx 0.2%
3x5' PBS-Tx 0.2%
Blockade Nonspecific Binding
60' incubation in:
- 10% Normal Goat Serum (Vector), 0.2% gelatin, 2% non-fat milk, in PBS-Tx-0.2%
1 day Primary Antibody Incubation (4 Degrees)
- Antibodies:
- 3% Normal Goat Serum (vector), 0.2% gelatin in PBS-Tx 0.2%
DAY 2
3x5' PBS-Tx 0.2%
Secondary Antibody Incubation (4 Degrees)
1-2h incubation in:
- 1:200 Goat anti-Rabbit (Mouse or rat) biotinylated antibody (Vector)
- 3% Normal Goat Serum (Vector), 0.2% gelatin, in PBS-Tx 0.2%
3x5' PBS-Tx 0.2%
*** ABC preparation 30-45 before use
Avidin-Biotin Complex (ABC Vector), (Room temperature, slow shaking)
60' incubation in:
- 1:300 ABC in PBS-Tx 0.2%
3x5' PBS-Tx 0.2%
1x 5¡¯ Tris buffer, 0.05M, ph 7.5
Development in diaminobenzidine (DAB)
DAB solution:
- one tablet of DAB (sigma D-4293) in 5 ml of water
Add to filtered DAB solution:
0.01% Hydrogen peroxide (3.3ul of 30% H2O2 per 10 ml DAB solution)
2x5' washes Tris Buffer,
dehydrate, immerse in Xilene and cover.
Or,
Secondary Antibody Incubation (4 Degrees)
1-2h incubation in:
- 1:300 Goat anti-Rabbit (Mouse or rat) fluorescent antibody (Molecular Probes)
- 3% Normal Goat Serum (Vector), 0.2% gelatin, in PBS-Tx 0.2%
3x5' PBS-Tx 0.2%
counterstain and cover
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