PLZF Antibody (D-9): sc-28319

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Testes were fixed for 2 h to overnight in 4% PFA, washed multiple times in 1× PBS, transferred to 30% sucrose, embedded in O.C.T, and stored at −80°C. Five µm thick sections were cut and placed on positively-charged slides and stored at −20°C prior to use. Immunolabeling was performed using standard methods. Briefly, blocking and antibody incubations were done at room temperature in 1× PBS containing 3% BSA + 0.1% Triton X-100, and stringency washes were done with 1× PBS +0.1% Triton X-100. Sections were blocked for 1 h prior to 1 h incubation in primary antibody (see Table 1). Secondary antibodies (1:500, Alexa Fluor-488 or -555, Invitrogen) plus phalloidin-635 (1:500, Invitrogen) were incubated for 1 h at room temperature. Primary antibody was omitted as a negative control. Coverslips were mounted with Vectastain containing DAPI (Vector Laboratories), and images obtained using a Fluoview FV1000 confocal laser-scanning microscope (Olympus America). Immunolabeling was performed in triplicate on testes from at least 2 different animals. ImageJ (U. S. National Institutes of Health) was used to assess images used for quantitation. Between 21–30 cords from at least 2 different animals were counted. Each marker was co-stained with DDX4 to label all germ cells. Cells where counted as positive for a marker when selected by the threshold tool using the default algorithm. Thresholds for each marker counted were as follows: STRA8 = 70–255, KIT =70–255, DDX4 = 100–255, ZBTB16 = 50–255, CDH1 = 100–255, GFRA1 = 50–255.

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