E63-1 Antibody Staining Of Salivary Glands

updated 12/07/05 - Benjamin Costantino

(For the PDF version, click here.)

Recipes:

PEM
60mM PIPES
5mM EGTA
5mM MgCl₂
pH 6.9

Procedure:

1. Make fresh reagents each time: PEM (50mls good for 4 wells), 4% paraformaldehyde (0.04g paraformaldehyde into 1mL PEM, heat 70°C until dissolved).

2. Dissect salivary glands in PBS or D-PBS and keep in buffer until ready for fixing (no longer than 15 min).

3. Transfer glands into glass slide with wells containing 200µl of 4% paraformaldehyde fix for no more than 15 min. room temp.

4. Transfer fixed glands into Corning Transwell (Corning #3472) insert containing .1ml of PEM in insert and .6ml in well (NOTE: this volume will always be used unless otherwise specified). Wash for 3x5 min., RT, light shaking.

5. Permeabilize with 1ml 0.4% Triton X-100 in PEM 3x10min. RT, light shaking.

6. Block in: 10% Normal Goat Serum/ 0.4% Triton X-100 in PEM [Block] - for 1hr. RT.

7. Incubate w/Anti-E63-1 in Block (1:10,000) overnight, 4°C on Orbitron.

8. Wash 3x10min PEM/Triton.

THE FOLLOWING STEPS MUST BE DONE IN THE DARK:

9. Incubate 2 hrs. with GAR-FITC in Block (1:200), RT.

10. Wash 2 x 10 min in PEM/Triton, RT. Then, 2x10min in PEM, RT.

11. Aspirate glands out of insert using P200 and transfer to glass slide with wells.

12. Transfer glands for mounting on slide with a drop of mounting solution (80% 1M Tris pH 8.8 + 0.5% PDA and 20% glycerol). Seal slide with wax or nail polish.