Golgi Immunostaining Of Salivary Glands
updated 02/24/06 - Benjamin Costantino
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Procedure
1. Make fresh fix solution each time: 4% paraformaldehyde and 1.5% glutaraldehyde in PBS. (0.04g paraformaldehyde and 15µl gluteraldehyde into 1mL PBS, 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 fix solution for no more than 15 minutes room temp.
4. Transfer fixed glands into Corning Transwell (Corning #3472) insert containing 100µl of PBS in insert and 600µl in well (NOTE: this volume will always be used unless otherwise specified). Wash for 3x5 min., RT, light shaking.
5. Permeabilize with 0.3% Triton X-100 in PBS 3x10min. RT, light shaking.
6. Block in: 5% Normal Goat Serum/ 0.3% Triton X-100 in PBS [Block] - for 1hr. RT.
7. Incubate w/ Anti-Golgi (mouse) in Block (1:250) overnight, 4°C on Orbitron.
8. Wash 3x10min 0.3% Triton-X in PBS.
THE FOLLOWING STEPS SHOULD BE DONE IN THE DARK:
9. Incubate 2 hrs. with GAM-FITC in Block (1:200), RT.
10. Wash 2x10 min in Triton/PBS, RT. Then, 2x10min in PBS, 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 pH8.8 + 0.5% PDA and 20% glycerol). Seal slide with wax or nail polish.
