Antibody Staining of Imaginal Discs

From DIS- Thom Kaufman's lab
 
1. Dissect tissues in Tri-PBS, gather in a 1.5 ml tube of Tri-PBS on ice(at least 10 animals can be processed in one tube).  Tear larvae in half and invert the anterior portion; remove all the gut tissue from the mutant larvae by cutting at the esophagus just anterior to the proventricus. Prepare control larvae as above, but leave the proventriculus attached to the esophagus to serve as marker, and process both in same tube.

2. Replace tri-PBS with 280 ul PBS, 120 ul 10% paraformaldehyde in PBS, and 500 ul heptane. Shake by hand for 30-45 seconds.

3. Replace first fixative with 520 ul of PBS, 240 ul 10% paraformaldehyde in PBS, and 40 ul of fresh DMSO; rock for 20 minutes.

4. Remove 2nd fix and wash twice with MeOH.

5. Replace MeOH wash  with 980 ul MeOH and 20 ul 30% hydrogen peroxide; rock for 30 minutes.

6. Wash 4 times in 1 ml of 0.1% BSA in Tri-PBS, 10 minutes each wash.

7. Replace last wash with 452 ul Tri-PBS, 5 ul 10% BSA,  and 40 ul normal goat serum (NGS); rock for 30 minutes.

8. Add primary antibody directly to the tube and rock overnight at room temperature.

9. Wash tissues 5 times in 1 ml 0.1% BSA in tri-PBS, for 5, 10, 15, 20, and 25 minutes, respectively.

10. Replace last wash with 445 ul Tri-PBS, 5 ul 10% BSA, and 40 ul NGS, rock for 30 minutes.

11. Add 10 ul HRP conjugate secondary antibody? and rock for 90 minutes.

12. Wash twice, 10 minutes each, in 0.1%BSA in Tri-PBS, followed by three washes in Tri-PBS.

13. Replace last wash with 450 ul Tri-PBS and 50 ul DAB 5 mg/ml in 0.1 M Tris pH 7.5; rock for 5 minutes. Add 5 ul of 0.3% hydrogen peroxide in water. Monitor reaction under dissecting scope; stop reaction by removing DAB solution and quickly washing tissue 5 times in PBS. Dissect discs and mount in Aqua-mount.

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Tri-PBS   pH 7.5:

137 mM NaCl

2.7 mM KCl

10.1 mM Na2HPO4

1.8 mM KH2PO4

0.2% Triton X-100