Doris Wu (4/06)
Whole-mount HCA staining
1) Dissect inner ears and puncture some holes in the membranous labyrinth if
necessary.
2) Pre-incubate with DMEM-10% FCS + 0.4% Triton (MFT) in 12-well plates for
several hours at room temp.
3) Incubate with anti-HCA antibody at1:5000 dilution in MFT at 4C for 24 hours.
4) Wash with PBS with 0.1% Triton (PBT) all day long and overnight.
5) Incubate with biotinylated anti-mouse (Vector Laboratories) at 1:500 dilution in
MFT for 24 hours.
6) Wash with PBT overnight at 4C.
7) Incubate with Streptavidin-AP (Zymed) at 1:500 dilution using SA dilution buffer
overnight at 4C.
8) Wash with PBT all day long at RT.
9) Wash with pre-detection buffer 3 times, 10-15 min each
10) Incubate with reaction mixture and check reaction every 5 min. Should take 10-15
min or so. The anti-HCA antibody usually has very little or no background.
11) Stop reaction in a PBT-EDTA solution (PBT + 1mM EDTA)
* All day-time incubation and washes are done at room temp and all overnights are done
in the cold room or refrigerator.
Solutions:
PBT
200 ml
20 ml
1780 ml
10xPBS
10% Triton
distilled water
SA Dilution buffer (0.1% BSA, 5 mM EDTA, 0.5 % Triton X-100, 1x PBS)
0.5 gm
BSA
5 ml
0.5 M EDTA
25 ml
10% Triton-X100
50 ml
10x PBS
420 ml
distilled water
Pre-detection buffer (100 mM Tris pH 8.8, 100 mM NaCl, 5mM MgCL2)
20 ml
1M Tris pH 8.8
4 ml
5 M NaCL
1 ml
5 mM MgCL2
Detection solution
50 ug
BCIP (5-bromo-4-chloro-3-indolyl phosphate)
5 mg
nitroblue tetrazolium (optional)
in 5 ml pre-detection buffer