Anti γ h2ax primary antibody

A total of 5 × 10⁴ cells were seeded into a confocal laser dish 1 day before irradiation with 8 Gy. Four hours post irradiation, cells were fixed with 4% paraformaldehyde at room temperature for 30 min and permeabilized with 0.1% Triton X-100 for 2 h. Cells were then blocked with 5% BSA for 90 min and washed with PBS. After incubation with the anti-γ-H2AX primary antibody (1:400; Cell Signaling Technology) overnight at 4 °C, cells were washed with PBS and incubated with an Alexa Fluor 555-conjugated secondary antibody (Beyotime) for 90 min. Cells were washed with PBS, treated with DAPI staining solution for 20 min and observed using a confocal fluorescence microscope (Leica).

Embryos: Following fixation, embryos were analysed by immunohistochemistry for DNA damage repair (anti-γH2AX primary antibody; Cell Signaling Technology, Danvers, MA, USA) and O-GlcNAc (pan O-GlcNAc RL2 antibody; Abcam, Melbourne, VIC, Australia) in three experimental replicates, with at least 15 embryos per replicate using published methods^{15 (link)}. Ovary, oviduct and uterus: 7 µm paraffin embedded tissue sections were analysed for glycosylation and stress deflection (RL2 and HSP90a: Abcam, Melbourne, VIC, Australia, Pan O-GlcNAc CTD110.6: (Sigma), GRP94 and HSPA5: Novusbio, Littleton, CO, USA) in six mice/treatment/time point (20 sections/mouse) using standard methods^{62 (link)}. Fluorescence was detected using a Fluoview FV10i Confocal Laser-Scanning Microscope (Olympus) and instrument settings were constant for each replicate. Images obtained were processed and analyzed using Fiji software (National Institutes of Health), with pre-developed plugins and macros. For embryos, regions of interest were whole cells and nuclei only, and cytoplasmic staining was the difference between these values; the blastocoel cavity was excluded from blastocyst analysis. For reproductive tract tissues, regions of interest were the oviductal epithelium and uterine epithelium (details; figure legends).

KYSE 150 cells
were cultured in glass-bottom dishes for 24 h, treated
with UiO-66-NH₂(Hf) (1.0 mL, 0 or 50 μg/mL) for 4
h, and exposed to X-ray irradiation (0 or 6 Gy). After 3 h, the cells
were fixed in 4% paraformaldehyde for 1 h, permeabilized with Triton
X-100 (0.5 vol %) for 5 min, incubated in normal goat serum (5 vol
%) for 1 h, and then incubated with an anti-γH2AX primary antibody
(1:400, Cell Signaling Technology, Cat# 9718) at 4 °C overnight.
The cells were washed three times with phosphate-buffered saline (PBS)
and incubated with a DyLight 594-labeled secondary antibody (1:500,
Invitrogen, Cat# 35560) for 1 h at room temperature. Finally, the
cell nuclei were counterstained with Hoechst 33342 (20 μM).
Laser scanning confocal fluorescence images were acquired. Cells treated
with neither UiO-66-NH₂(Hf) nor X-ray irradiation were
used as controls.