Wholemount antibody staining - alkaline phosphatase detection (Conlon lab)

Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

Whole-Mount Antibody Staining Protocol

1) Fixation

a. Remove vitelline membranes and fix in MEMFA 1.5-2 hrs.

b. Replace with 100% EtOH a few times (Store @ -20°C)

2) Rehydration

a. 75% MeOH/25% PBST (PBS + 1% Triton [permeabilizes cellular and nuclear membrane] or Tween [permeabilizes cellular only])

b. 50% MeOH/50% PBST

c. 25% MeOH/75% PBST

d. 3X in PBST

3) Blocking

a. PBST + 10% Serum + 0.1% BSA

b. 1hr (minimum) @ RT

4) Primary Ab Incubation

a. Blocking solution + diluted antibody

b. ON @ 4°C

5) Washes

a. 5X 1 hr @ RT w/ PBST

6) Blocking – same as step # 4

7) Secondary Ab incubation

a. Same as step # 5 with appropriate antibody.

8) Washes

b. 5X 1 hr @ RT w/ PBST

9) Alkaline Phosphatase Staining

a. Dissolve one DAB tablet in 30 mL PBS

b. Add 20 mg Nickel Chloride

c. Add 1-2 mL of staining solution to each vial

d. Incubate in DARK for 30 min.

e. Add 1 μL/mL 30% H2O2

f. Develop in dark (10-30 min.)

g. Rinse 3X in PBS

h. Fix in MEMFA for 30 min.

i. Rinse w/ PBS