Immunofluorescence

Larval Brain Fixation (in microwell plate)

Required Solutions:

- Schneider’s medium

- fixative: 4% paraformaldehyde in PEM buffer plus 0.1% Triton X-100

- wash buffer: PBT (1xPBS, 0.1% Triton X-100)

- blocking buffer: PBT plus 10% Normal Goat Serum (NGS)

- 1˚ antibodies

- fluorescently conjugated 2˚ antibodies (directed against 1˚ antibodies)

Procedure:

1. Raise animals in un-crowded conditions:

- Place adults (males and females) into a collection bottle on a grape cap with freshly prepared yeast paste (make sure everything is pre-warmed).

- Let flies lay for desired period of time.

- Take plate with embryos off collection bottle and put plate for 22 hours in a 25˚ incubator.

- Next day, pick 50-60 L1 larva/med vial, 20 L1 larva/small vial, or 30/small Petri dish (35mm) with a probe under the dissecting scope (select desired genotype using visible markers if possible).

2. Let animals develop, pick larvae at desired developmental stage.

3. Dissect larva in Schneider’s medium (or D22 if Schneider’s is not available) and transfer brain with cuticle still attached into 0.5ml tube with fixative. Fix for 20mins on nutator at RT. Fixation time may need to be adjusted depending on antigen-antibody combination.

4. Rinse 3x quickly with PBT.

5. Wash 4x15min with PBT, keep tube on nutator between washes

6. Replace wash buffer with blocking buffer, incubate on nutator for 1h at RT or o/n at 4˚C

7. Transfer brain to glass depression well block, dissect brain, transfer brain to microwell plate (up to 5 brains/well)

8. Replace blocking buffer with 1˚ antibody solution (1˚ antibodies in blocking buffer). Use 10ul/well. Wrap plate in parafilm and incubate at 4˚C o/n.

9. Wash brains 3x quick with PBT, then 4x15 min with PBT, and 1x15min in blocking buffer.

10. Replace blocking buffer with 2˚ antibody solution (1˚ antibodies in blocking buffer). Use 10ul/well. Wrap plate parafilm and cover with aluminum foil. Incubate 2h at RT or o/n 4˚C.

11. Wash brains 3x quick with PBT , wash brains 5x10min with PBT at RT, keep plate covered with aluminum foil between washes

12. Put antifade on, rewrap plate with parafilm, incubate overnight 4˚ (keep in dark). Mount brains on object slide with coverslip (size 1.5 for 3rd instar larvae) bridge (keep in dark).

13. IMAGE!!!!!!