Sc 22839

Whole-mount immunofluorescence of seminiferous tubules was performed as described (Carrieri et al., 2017 (link)). For the expression analysis of TAG-NANOS2 and GFRα1, co-staining of tubules was done with two primary antibodies: α-GFP, 1:500 (GFP-1010, Aves), α-GFRα1, 1:50 (GT15004, Neuromics). For the expression analysis of TAG-NANOS2 and c-KIT, co-staining was done with: α-GFP, 1:200 (A-11122, ThermoFisher) and α-C-kit, 1:250 (AF1356-SP, R&D Systems). For the expression analysis of TAG-NANOS2 and PLZF: α-GFP, 1:500 (GFP-1010, Aves), α-PLZF, 1:100 (sc22839, Santa Cruz Biotechnology). For the expression analysis of TAG-NANOS2 and Dcp1a: α-GFP, 1:500 (GFP-1010, Aves), α-Dcp1a 1:500 (WH0055802M6, Sigma-Aldrich). Secondary antibodies used were all Invitrogen: goat anti-Chicken, Alexa Fluor 488 (A-11039); donkey anti-Rabbit, Alexa Fluor 488 (A-21206); donkey anti-goat, Alexa Fluor 568 (A-11057). Images were acquired by using a Confocal microscope, by taking Z-stacks, and with Z-stepsize set at 0.34 μm. Images were analyzed with Fiji ImageJ. A_s and A_pr spermatogonia were distinguished by using the 25 μm topographical criteria (Huckins, 1971 (link)). If the internuclear distance between two spermatogonia was over 25 μm, cells were assigned to the A_s category. On the contrary, spermatogonia whose internuclear distance was smaller than 25 μm, were considered to belong to the same chain.

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).

Human thyroid tissues were homogenized in 300 μl of RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate in PBS with a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN)) and centrifuged at 15000 rpm for 20 min at 4°C. The supernatants were used as the total cell lysates. A aliquot (10 μg protein) of each lysate was subjected to 10% SDS-PAGE, and the separated proteins were electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline containing 0.05% Tween-20 and 5% non-fat dried milk, washed, and incubated with polyclonal primary antibodies against PLZF (sc-22839; Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:200 dilution) and β-actin (sc-1616; Santa Cruz Biotechnology Inc.; 1:500 dilution). The membranes were then exposed to an anti-rabbit secondary antibody (NA934; Amersham Pharmacia Biotech, Little Chalfont, UK; 1:5000 dilution). After incubation with the secondary antibody, detection of the bound antibodies was performed using enhanced chemiluminescence.