Fig2a P5 STRA8 and DDX4 whole mount co-IF

Raw image dataset for P5 seminiferous tubules STRA8 and DDX4 whole mount co-immunofluorescence (Figure 2a)<br>Please see Hobbs, R. M.<i> et al.</i> Distinct germline progenitor subsets defined through Tsc2-mTORC1 signaling. <i>EMBO Rep</i> <b>16</b>, 467-480, doi:10.15252/embr.201439379 (2015) for protocol. <br>P5 seminiferous tubules were fixed in 4% PFA for 6hrs at 4C, washed 3 times in PBS and blocked in blocking buffer (20% FBS, 0.3% Triton X in PBS) for at least 2 hours at 4C. Then tubules were incubated with primary antibodies (rabbit anti-STRA8 (abcam ab49405) 1:200 and mouse anti-DDX4 (Abcam ab27591) 1:500 diluted in blocking buffer) overnight at 4C. This was followed by 6 PBS washes and incubation with secondary antibodies (goat anti-rabbit Alexa 555 (Invitrogen A21428) 1:250, goat anti-mouse Alexa 488 (Invitrogen A11001) 1:250 and DAPI 5mg/ml (Sigma D8417) 1:10,000 diluted in blocking buffer) for 4 hours at 4C. Tubules were washed 6 times in PBS and mount in 70% glycerol. STRA8 and DDX4 co-staining/z-stacked images were captured on Leica SP8 inverted confocal microscope (Leica Microsystems).