Histology and immunohistochemical analysis were done as described by us in [12 (link)]. Briefly, tissues were overnight fixed at 4°C, in 4% PFA, embedded in paraffin and 5 μm sections were prepared. Slides were deparaffinised and incubated with primary antibodies including βcatenin (610154, BD Transduction Labs, CA, USA); PCNA, Plzf (sc-56: PCNA; sc-22839: Plzf; Santa Cruz Biotechnology, CA, USA); Foxo1, LEF1, TCF1 (#2880: Foxo1; #2230: LEF1; #2203: TCF1; Cell Signaling Technology, MA, USA); Cyclin D1, SCYP3, Stra8 (ab16663: Cyclin D1; ab15093: SCYP3; ab49602: Stra8; Abcam, Vic, Australia); GCNA [49 (link)]; γH2AX (#05-636: γH2AX; Millipore, MA, USA), αSMA (c6198, Sigma, MO, USA) and AlexaFluor secondary antibodies (1:250; Jackson ImmunoResearch Labs, PA, USA). For detection of apoptotic cells, TUNEL assay was performed on paraffin sections as per the instructions provided with the kit (Millipore). For cell counting images at 20x magnification were taken with Olympus DP72 microscope keeping same exposure and gain for both control and mutant tissues. Each testis was divided into four sections and at least two images were randomly selected from each section from minimum three control and mutant animals. The cells were counted using ImageJ (National Institute of Health, USA).
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