Antibody Staining of Larval Imaginal Discs

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

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