Anti ph3 ser10

The commercial primary antibodies in this paper were as follows: anti-REPO (8D12, DSHB); anti-pH3 (Ser10) (H0412, Millipore); anti-Cleaved-Caspase 3 (9664, Cell Signaling); anti-α tubulin (ab18251, abcam); anti-MEK (8727, Cell Signaling); anti-p-MEK (Ser217/221) (3958, Cell Signaling); anti-ERK (sc-514302, Santa Cruz); anti-p-ERK (T202/Y264) (4370, Cell Signaling); anti-KCNMA1 (sc-374142, Santa Cruz); anti-Ki-67 (9129, Cell Signaling); and anti-GAPDH (ab8245, abcam).
Three BRAF-related antibodies were used in this study: anti-BRAF (sc-5284, Santa Cruz) were used to detect the total levels of BRAF; anti-BRAF^V600E specifically recognizing mutated amino acid sequence (ab228461, abcam) were used to detect the levels of BRAF^V600E; anti-p-BRAF (S729) specifically recognizing phosphorylated site (ab124794, abcam) were used to detect the levels of BRAF with serine 729 phosphorylation, to measure the BRAF activation levels regardless of the presence of BRAF mutations.

Tissues were fixed overnight either in 4% PFA or in Bouin's fixative and embedded in paraffin. Five-mm sections were stained with haematoxylin and eosin (H&E) or processed for immunofluorescence (IF). For IF analysis, PFA-fixed sections were incubated overnight at 4°C with the following primary antibodies: anti- GATA4 (Santa Cruz Biotechnology, sc-9053, 1∶500), anti-protamine 1 (Shal technologies, Hup1N, 1∶1000), anti-pH3 (Ser10) (Millipore, Cat#06-570, 1∶500), anti-γH2AX (Calbiochem, Cat#dr-1017, 1∶500) and anti-H3K9me3 (Millipore, Cat#07-523, 1∶500). Alexa-conjugated secondary antibodies (Invitrogen) were then used for signal revelation and sections were counterstained using DAPI. All images were obtained with a Zeiss Axioscope microscope and processed using the ZEN software.

For histological analyses, testes were fixed for 24 h at room temperature (RT) in Bouin’s solution. Tissue was further paraffin-embedded, sectioned (7 μm), and stained with H&E. For immunohistology, testes were essentially fixed in 4% paraformaldehyde for ∼24 h. Paraffin-embedded tissues were further sectioned at 7 μm. For TUNEL and PH3 double immunostaining, sections were deparaffinized followed by antigen retrieval using Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0). Sections were blocked in PBS containing 5% goat serum for 1 h at RT, followed by TUNEL staining, performed following instructions manufacturer’s instruction (Invitrogen, Click-iT™ TUNEL kit). This was followed by 1° antibody incubation overnight at 4 °C (anti-pH3 (Ser10), 1:100, Millipore) and anti-rabbit AF488 (Invitrogen, 1:1000) 2° antibody incubation for 1 h, RT. Slides were counterstained using DAPI, mounted in Vectashield and imaged as described in below.