Ab64965

Testes were fixed at 4° C in Bouins solution, and dehydrated for paraffin embedding. Sections were cut to a thickness of 5 μM, deparaffinized and rehydrated. For histology, sections were stained with hematoxylin and eosin (H+E). For immunohistochemistry, slides were prepared by performing antigen retrieval. Slides were boiled in 0.01 M sodium citrate, pH 6.0, for 10 min. Sections were then blocked for 1 h at room temperature with 3% goat serum in PBS. Primary antibodies were diluted in 3% goat serum in PBS and incubated with sections overnight at 4° C. Primary antibodies used for IHC were anti-SYCP3 (#ab15093, Abcam, Cambridge, MA), anti-γH2AX (#05-636, Millipore, Billerica, MA), anti-STRA8 (#ab49602, Abcam), anti-MED1 (#ab64965, Abcam), anti-TRA98 (#ab82527, Abcam), anti-BOULE (gift of Eugene Xu, Northwestern University), anti-SOHLH1 (gift of Aleksandar Rajkovic, University of Pittsburgh). Sections were then labeled with the appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. Slides were incubated for 10 min with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were then affixed with Vectashield anti-fade mounting medium (Vector Laboratories, Burlingame, CA) and samples were imaged using a Leica DMR-HC epifluorescence microscope with 10x, 20x, and 40x objectives and a QImaging Retiga 4000R camera.

Murine ESCs were plated onto glass coverslips in preparation for immunostaining. After 24 hours, cells were fixed with 4% paraformaldehyde in PBS, washed 3 times with PBS, and then permeabilized with 0.5% triton-X100 in PBS for 10 min at room temperature. Cells were then washed 3 times with PBS, blocked for 1 hour in 4% IgG-free BSA (Jackson ImmunoResearch 001-000-162) in PBS, and then stained over night with the indicated antibody in 4% IgG-free BSA at room temperature in a humidified chamber. Cells were then washed 3 times with PBS. Secondary antibodies were added to cells in 4% IgG-free BSA and incubated for 1 hour at room temperature. Cells were then washed twice in PBS. Cells were stained with Hoechst dye (Molecular Probes H3570) in PBS or water for 5 minutes, and then mounted in Vectashield mounting media (Vector Laboratories H-1000). Imaging was performed on an RPI spinning disk confocal at 100X magnification using the MetaMorph software v. 7.10.3.279 (Molecular Devices). Primary antibodies: anti-MAP2 (Invitrogen MA5-12823) and anti-MED1 (Abcam ab64965). Secondary antibodies: anti-mouse Alexa Fluor 568 (Invitrogen A11031) and antirabbit Alexa Fluor 555 (Invitrogen A21428).