Whole Mount Antibody Staining
Fix embryos in 4% p-formaldehyde in PBS buffer for 3 to 6 hours at room temp or overnight at 4 degrees C. (some antibodies are sensitive to too-long fixation).
Wash in 0.1 M PO4 buffer 5 min.
Wash in DW 5 min.
Freeze in acetone at -20 C. 7 min. to permeabilize the tissue (or permeablize with proK as for RNA in situ).
Wash in DW 5 min.
Wash in 0.1 M PO4 buffer 5 min.
Block with PBDT (PBS/1% BSA/1% DMSO/0.1% Triton) containing 2% normal goat serum to block non-specific binding sites for 30 min at RT.
Treat with primary antibody (eg. mouse anti-fish)diluted appropriately in block for 5 hours at RT or overnight at 4 C.
Wash 4X (10,15, 30 and 60 min ) in PBDT (no goat serum).
Treat with secondary antibody (eg. Biotinylated goat anti-mouse) in PBDT overnight at 4 C or for 5 hours at RT.
Wash 4X (10,15, 30 and 60 min. each) in PDT (PBS/1% DMSO/0.1% Triton).
Prepare ABC reagent: add 10?l reagent A and 10?l reagent B to 1ml PDT.
Incubate A and B reagents together for 30 min before adding to embryos.
Treat embryos for 2 hours at RT with ABC reagent.
Wash 5X (10, 15 and 3x30 minutes each) in PDT. Can leave O/N at 4 C at this point.
Stain for HRP using DAB
DAB is a carcinogen. Wear gloves, change gloves frequently. Work on a piece of bench-cote inside the hood. Decontaminate all materials with bleach after staining is complete. Store liquid waste in bottle marked "DAB waste" in hood. If you bring your embryos out of the hood to look at them under the stereomicroscope, put them on a piece of benchkote on the stage of the microscope, and change your gloves before touching the focus control.
Prepare two 5-ml snap cap tubes, each containing 4 mls 0.05M PO4 buffer, pH 7.4, 1% DMSO and 50 ?l DAB (stock solution is 40mg/ml). One of these is the presoak. To the other one, add 5 ?l 3% H2O2. This is the staining solution.
- wash embryos once in 0.05M PO4 buffer, pH 7.4 containing 1% DMSO
- presoak embryos, 5 min.
- stain embryos by removing presoak and adding staining solution. Monitor by visualizing under a stereomicroscope, but minimize exposure to bright light as this will cause higher background staining. Reaction time is usually in the range of 1 to 20 minutes.
- Stop reaction by adding 0.1M PO4, 1%DMSO. Wash in DW, several changes.
- Store in PBST or PBST/Glycerol
Embryos may be mounted in 70% glycerol, or dehydrated and mounted in permount.
For mounting in Permount:
-Dehydrate tissue through an ethanol series (50%, 70%, 85%, 95%, and
absolute X2, 5 minutes each), clear in methyl salicylate. Mount tissue in
Permount between two coverslips, using strips of teflon tape or pieces of glass at each end to act as spacers. The tissue may also be stored in methyl salicylate and viewed using depression slides.
