Vectashield mounting medium with 4 6 diamidino 2 phenylindole

Cultures were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 15 min at room temperature and then blocked in 0.5% bovine serum albumin for 1 h. Primary antibodies specific for acetylated α-tubulin (T7451, Sigma) and γ-tubulin (T6557, Sigma) were added apically in 0.5% bovine serum albumin and incubated for 1 h at room temperature. After washing with PBS, fluorochrome-conjugated secondary antibodies (Alexa Fluor, Life Technologies) were added for 1 h at room temperature. Finally, transwell filters were washed in PBS and mounted on slides using Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector labs). Images were acquired either on a Zeiss LSM 710 Laser Scanning microscope ( × 20 and oil immersion × 63 lenses) using the ZEN software for image analysis or on an Olympus VS120 slide scanner. Measurements of acetylated α-tubulin staining were made using regions of interest that cover the whole transwell. Binary images were generated by global thresholding and measured using the ImageJ 1.4j application.

Apoptosis of the ovarian follicles was detected using an In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) as previously described by us [31 (link)]. OTs were mounted using VECTASHIELD Mounting Medium with 4',6-diamidino-2-phenylindole (Vector Laboratories, California, USA) and visualized under an inverted Zeiss AX10 fluorescence microscope (Carl Zeiss, Oberkochen, Germany); cells with fragmented DNA displayed green fluorescence and normal cells, which were counterstained, displayed blue fluorescence. Follicles with over 30% apoptosis-positive cells (emitting a green signal) were categorized apoptotic follicles [12 (link), 31 (link)–33 (link)].

MCF-7 cells were rendered quiescent by serum starvation and subsequently stimulated with serum for 6 h and 18 h. The cells were stained as per the protocol described previously [23] (link). Double immunofluorescence experiments were conducted using rabbit anti-human KDM2A (1∶200 dilution) and mouse anti-human E2F1 (1∶200 dilution) antibodies. Secondary antibodies were goat anti-rabbit Alexa Fluor 555 (1∶1000 dilution) and goat anti-mouse- Alexa Flour 488 (1∶200 dilution). The cells were mounted with VECTASHIELD mounting medium with 4′ 6-diamidino-2- phenylindole (Vector Laboratories). Cells were observed using a Leica TCS SP5 confocal microscope (Leica Microsystems) at 630× magnification.

Cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.3% Triton X-100 in PBS for 10 min, and blocked with 5% normal goat serum in PBS for 1 h at room temperature. The cells were incubated with primary FANCD2 antibody (Cell Signaling Technology) for 1 h, washed three times with PBS, and then incubated with Alexa Fluor 488-Anti- and Texas Red-conjugated secondary antibodies for 1 h (Invitrogen). Then, cells were washed three times with PBS and mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories).

Wheat germ agglutinin was used to stain cardiomyocyte cell borders at all time points using a previously described method [Texas Red-X conjugate, (Molecular Probes, Life Technologies)] (13 (link)). Hearts were mounted onto a chuck in the orientation of the heart in vivo using Tissue Tek optimal cutting temperature compound (Sakura). The heart was sectioned in the coronal plane at −21 C until reaching the mid-cardiac region. Two 7-μm sections were cut consecutively and mounted onto a charged slide. Sections were fixed in 10% NBF for 20 minutes at room temperature and washed with phosphate buffered saline (PBS) before incubating with 10 μg/ml of conjugated agglutinin while gently rocking in darkness for 2 hours at room temperature. Slides were then washed with PBS and air-dried before addition of Vectashield mounting medium with 4′,6-Diamidino-2-phenylindole (Vector Laboratories). Images were analyzed double-blinded using CellD software (Olympus). Only myocytes in cross-section were selected. Area was calculated by averaging across five separate images per heart (approximately 400–600 cells measured per heart).