Antibody Staining of Larval Imaginal Discs

1. Dissect discs in PBS.

2. Fix in 3% formalin in PBS for 15-30 minutes at room temperature.

3. Wash for 30 minutes in 3 changes of PBT.

4. Incubate with primary antibody in PBTN for 1 hr. to overnight. 

5. Wash tissue for 30 min. to overnight in PBS.

6. Incubate for 1 hr. to overnight with HRP-conjugated secondary

    diluted 1:100 in PBTN.

7. Wash 45 min. with 3 changes of PBS.  

8. Incubate in 200 ul of 1mg ml-1  diaminobenzidine in 0.12 M Tris,

    pH 7.6 for 10 min. 

9. Add hydrogen peroxide to a final concentration of 0.0015% and

    allow the stain to develop. 

10. Stop reaction by washing 2X in PBT.

11. Mount under a coverslip in 90% glycerol, 0.1 M Tris, pH7.6. 

PBT

PBS

0.2% BSA

0.1% Triton X-100

PBTN

PBT

2% normal goat serum

Jack, et al. 1991. Development 113: 735-47.