Jun 26, 2024
Version 1
Immunofluorescence staining on larval and adult Drosophila gonads V.1
- Samantha Goetting1
- 1Johns Hopkins University
Protocol Citation: Samantha Goetting 2024. Immunofluorescence staining on larval and adult Drosophila gonads. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnd7ngk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 26, 2024
Last Modified: June 26, 2024
Protocol Integer ID: 102463
Abstract
Immunofluorescence staining protocol for Drosophila gonads
Materials
1x PBS
0.3% PBTx (0.3% Triton-X in PBS)
1% PBTx (1% Triton-X in PBS)
Paraformaldehyde or formaldehyde
Normal serum (usually NGS)
Primary antibodies
Secondary antibodies
DAPI
Before start
All steps are done with gentle rotation.
Day 1
Day 1
2h 40m
Dissect tissue in 1x PBS. Transfer to a 1.5 mL tube containing 1x PBS. If it is a quick dissection (<20 mins), no ice needed. If more time is needed, keep samples on ice.
Remove PBS and add fixative. Fix in 4% paraformaldehyde in 0.3% PBTx for 20 min RT with gentle rotation.
500 uL fixative = 125 uL of 16% paraformaldehyde + 375 uL 0.3% PBTx
20m
Aspirate the fixative and wash twice for 10 min in 1% PBTx. Not getting rid of fix will affect your immunostaining.
20m
Aspirate the supernatant and block/permeabilize for at least 2 hours in 1% PBTx + 5% normal serum, or overnight at 4°C.
1 mL block solution = 50 uL NGS + 950 uL 1% PBTx
2h
Primary antibodies are diluted in 0.3% PBTx + 5% normal serum and incubate for 1 hour at RT or overnight at 4°C. Overnight will give better staining.
Primary antibody mix in 0.3% PBTx + 15 uL NGS (300 uL total)
12h
Day 2
Day 2
5h 20m
Remove the primary antibody mix and wash in 0.3% PBTx three times for 20 min at RT.
1h
Wash in 0.3% PBTx + 5% normal serum twice for 30 min at RT.
1 mL wash solution = 50 uL NGS + 950 uL 0.3% PBTx
1h
Secondary antibodies are diluted in 0.3% PBTx + 5% normal serum and incubated for ~2 hours at RT or overnight at 4°C. Keep tubes covered from light.
2h
Aspirate the supernatant and add 500 uL DAPI to each tube. Incubate for 10 min at RT. Keep tubes covered from light.
10m
Aspirate the supernatant and wash in 0.3% PBTx three times for 20 min at RT.
1h
Aspirate the supernatant and wash in PBS for 10 min at RT.
10m
Store in PBS at 4°C or proceed with mounting. Keep tubes covered from light.
Protocol references
Slaidina et al. (2020)