Immunofluorescence staining on larval and adult Drosophila gonads

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 

Aspirate the fixative and wash twice for 10 min in 1% PBTx . Not getting rid of fix will affect your immunostaining.

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

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)

Remove the primary antibody mix and wash in 0.3% PBTx three times for 20 min at RT.

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

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.

Aspirate the supernatant and add 500 uL DAPI to each tube. Incubate for 10 min at RT. Keep tubes covered from light.

Aspirate the supernatant and wash in 0.3% PBTx three times for 20 min at RT.

Aspirate the supernatant and wash in PBS for 10 min at RT.

Store in PBS at 4°C or proceed with mounting. Keep tubes covered from light.