Immunostaining larval body wall neurons in Drosophila

This protocol has been used to for immunostaining the C4da neurons on the body wall of Drosophila larvae at multiple developmental times in the second and third instars. 

4% fixative solution

10ml 16% formaldehyde

75ml 1x PBS

0.2% PBS-T

1ml of 10% Triton X-100

49ml 1x PBS

0.2% Blocking Solution 

19ml PBST

1ml goat serum (Jackson Labs, 005-000-121) 

DAPI stain (1:10,000)

1ul DAPI 

10ml 1x PBS

Primary antibody (1:200)

in 0.2% Blocking Solution

• EcR-common DDA2.7, EcR-A 15G1a, or EcR-B1 AD4.4 (Developmental Studies Hybridoma Bank)

Secondary antibody (1:500)

in 0.2% Blocking Solution 

• Alexa Fluor 555 (Invitrogen A-2142A)

Day 1

1) Dissect larvae fillets in PBS on ice. Prepare larvae by pinning with 0.10mm insect pins in a Sylgard dish and cutting along the ventral midline. Remove internal organs and fat body and pin the body wall flat. Keep fillets pinned throughout staining.   

2) Fix for 10 min on shaker at room temperature in 4% fixative solution. 

3) Rinse with 0.2% PBS-T. 

4) Block for 2 hrs at room temperature with 0.2% Blocking Solution. 

5) Remove blocking solution. Add primary antibody solution and incubate overnight at 4^oC on rotoshaker. 

Day 2

6) Rinse with 0.2% Blocking Solution 3x in a row. 

8) Incubate with secondary antibody solution for 2 hrs at room temperature on rotoshaker.

9) Rinse with 0.2% PBS-T. 

10) Rinse with 0.2% PBS-T 10min (2x).

11) Stain with DAPI for 10 min at room temp.

12) Mount in Vectashield (Vector Laboratories) on microscope slides.