Anti fade mounting solution

For probing lung hypoxia, pimonidazole (20 mg/kg in PBS; Hypoxyprobe) were administrated via intraperitoneally into mice 1 hour before euthanasia. Followed by tissue processing and paraffin embedding, 5-μm-thick sections were cut. Slides were then dewaxed in xylene, rehydrated, and subjected to antigen retrieval by citrate buffer [10 mM trisodium citrate with 0.05% Tween 20 (pH 6.0)] at 95°C for 10 min. PBS-rinsed slides were permeabilized with 1% BSA with 0.1% Triton X-100 solution (PBS as diluent) for 15 min and blocked in 10% goat serum (Equitech-Bio) with PBS for 30 min at room temperature. Slides were hybridized with primary antibody, anti-Mab1 (1:50), overnight at 4°C in dark, followed by PBS wash for three times and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 5 μg/ml; Invitrogen). After washing, slides were mounted on the Antifade mounting solution (Vectashield), cover-slipped, and imaged on a Leica TCS SP8X STED3x super-resolution microscope. Hypoxic signals were quantified by ImageJ.

Apoptosis in the ovaries was assessed by TUNEL assay, which was performed by In
Situ Cell Death Detection kit (Roche, Basel, Switzerland). Paraffin sections of
ovaries were deparaffinized and washed twice with PBS and then stained with a
TUNEL reaction mixture. After washing twice with PBS, the sections were
counter-stained with DAPI. After washing twice with PBS, the stained sections
were mounted with anti-fade mounting solution (Vectashield, Burlingame, CA, USA)
and observed under a fluorescence microscope (Zeiss, Oberkochen, Germany).