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Last updated: 2/24/06 Benjamin Costantino Golgi Immunostaining of Drosophila Larval Salivary
 

Summary: Last updated: 2/24/06 Benjamin Costantino
Golgi Immunostaining of Drosophila Larval Salivary
Glands (Whole Mounts)
PROTOCOL:
1. Make fresh fix solution each time: 4% paraformaldehyde and 1.5% glutaraldehyde in
PBS. (0.04g paraformaldehyde and 15ul gluteraldehyde into 1mL PBS, heat 70C until
dissolved)
2. Dissect salivary glands in PBS or D-PBS and keep in buffer until ready for fixing (no
longer than 15min.)
3. Transfer glands into glass slide with wells containing 200ul of fix solution for no more
than 15min. room temp.
4. Transfer fixed glands into Corning Transwell (Corning #3472) insert containing 100ul
of PBS in insert and 600ul 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, 4C on Orbitron.
8. Wash 3x10min 0.3% Triton-X in PBS.
9. Incubate 2 hrs. with GAM-FITC in Block (1:200), RT. * **This and the following
steps must be done in the dark***

  

Source: Andres, Andrew - School of Life Sciences, University of Nevada at Las Vegas

 

Collections: Biology and Medicine