Whole mount immunostaining

Whole mount staining protocol for mouse seminiferous tubules.

The seminiferous tubules were teased apart in PBS using 5-Number forceps. 

The tubules were washed with PBS 3times and fixed in 4% PFA in PBS at 4 °C for 3-4hrs if staining for GFRA1. (Time varies with antibodies used)

After 4hrs the tubules were washed in PBS 3 times for 10 min each (no permeabilization needed for GFRA1 antibody). (Might require permeabilization for nuclear proteins, 15min incubation in 0.1% Triton-X 100)

The tubules were then incubated in 5% BSA (Sigma # A9647) in PBS (no detergent) for 1hr at RT. 

The tubules were incubated with Anti-GFRA1 antibody (1:250) in 5% BSA-PBS overnight at RT (the GFRA1 antibody works better at RT than 4 °C).

Next day the tubules were washed 3 times for 10 min each in 0.1% Triton-X 100, 1% BSA in PBS.

The tubules were then incubated with secondary antibody (1:1000) conjugated to Alexa- fluorophore for 1 hr at RT. 

Tubules were again washed in 0.1% Triton-X100 in PBS 3times for 10min each.

Tubules were then mounted in mounting media from Vectashield without DAPI and imaged on Confocal microscope.