Ab31333

Immunofluorescent staining was performed on 4 µm formalin-fixed, paraffin-embedded ovarian tumour sections. After deparaffinization, antigen retrieval was performed by boiling slides in sodium citrate buffer, and permeabilization was performed for nuclear antigens only using 0.25% v/v Triton X-100. Non-specific antibody binding was blocked with 5% BSA in PBS, and primary antibodies were applied and incubated overnight at 4 °C. Primary antibodies used were rabbit anti-wide-spectrum cytokeratin (#ab9377, 1:100, Abcam, Cambridge, UK), rabbit anti-Wilms tumor protein-1 (#ab89901, 1:100, Abcam), goat anti-PD-L1 (#AF1019, 1:125, R&D Systems, Minneapolis, MN, USA) and rabbit anti-p53 (#ab31333, 1:100, Abcam). Alexa Fluor® 594 or 647-conjugated goat anti-rabbit or donkey anti-goat secondary antibodies (1:1000, Abcam) were applied at 1:1000 for 1 h, and slides were mounted using ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher Scientific). Fluorescence images were captured using the Cytation 3 imaging Multi-Mode Reader (BioTek) and processed using the Gen5™ software v 2.05 (BioTek).

Mice were anaesthetized by using 2.0% isoflurane after connecting to a ventilator. After the left ventricle was perfused with phosphate-buffered saline (PBS) and paraformaldehyde (PFA), the heart was further fixed for 4 h in 2% PFA at 4 °C, followed by cryoprotection by immersion in 30% sucrose solution overnight before freezing in optimal cutting temperature (OCT) solution. Immunofluorescent staining of frozen sections (7 µm) was performed using primary antibodies to vascular endothelial cadherin (VECAD, ab33168), CD31 (ab28364), von Willebrand factor (vWF, ab11713), endothelial nitric oxide synthase (eNOS, ab5589), occludin (ab31721, all from Abcam, Cambridge, UK), Tek (AF762, R&D Systems, Minnesota, MN, USA), isolectin B4 (B-1205, Vector Labs, Burlingame, CA, USA), p53 (ab31333, Abcam), and associated fluorescein-conjugated secondary antibodies following the manufacturer instructions. Labelled sections were probed, and images were captured using a Leica TCS SP8 confocal microscope. Then, 8–10 fields were randomly chosen from each section of the heart, images were captured at ×63 magnification using a confocal microscope, and were each defined as one area. Then, co-localization analysis of confocal images was performed using the ImageJ software (National Institutes of Health [NIH]). Masson’s trichrome staining was performed on heart sections as described previously.

The antibodies for BRCA1 (OP92), γ‐H2AX (05‐636) and Vinculin (MAB3574) were purchased from Millipore (Quincy, MA, USA). The antibodies for RAD51 (ab63801), p53 (ab31333) and ATM (ab32420) were purchased from Abcam (Cambridge, MA, USA). Anti‐p21 (#2974), anti‐p‐CHK1 (S345) (#2348) and anti‐PARP1 (#9542) were obtained from Cell Signaling Technology (Danvers, MA, USA). All of the Alexa Fluor‐conjugated and HRP‐conjugated secondary antibodies were purchased from Molecular Probes (New York, NY, USA). Western blot analysis was performed as previously described,¹³, ⁴⁷ and the signal intensity was measured by ImageJ software (http://rsb.info.nih.gov/ij/).

Femurs were fixed in 10% formalin, decalcified in 10% EDTA for 4 weeks, and dehydrated using graded alcohol series and xylene, and processed for paraffin embedding and sectioning. Sections (7 µm) were stained with H&E and TRAP (Sigma, 387A‐1KT). For immunostaining, sections were treated with 3% hydrogen peroxide to block the endogenous peroxidase. The antigen retrieval was done by antigen unmasking solution following the instruction provided by the manufacturer (H‐3300; Vector Laboratories, CA, USA). The sections were incubated with 5% bovine serum albumin for 30 min at room temperature, then with primary antibodies to Cathepsin K (1:200, #ab19027, abcam), p16 (1:100; #PA30670, Invitrogen), p53 (1:100; # ab31333, Abcam), FGF23 (1:100; # MAB26291, R & D systems), SOST (1:12; # AF1589, R & D systems), overnight at 4 °C. Incubation with HRP‐conjugated secondary antibody (Cat. No. PK4001, Vector Laboratories, CA, USA) was done for 1 hour at RT. Immunoreactivity was detected using the horseradish peroxidase‐3,3′‐diaminobenzidine system (SK‐4100 vector laboratories, USA) followed by counterstaining with hematoxylin (Sigma). The images were acquired by Aperio CS2 Scanner (Leica Biosystems) and analyzed by Fiji ImageJ (version 1.51r; NIH).

MET, hydroxychloroquine (HCQ) and rapamycin (RAPA) were purchased from Sigma‐Aldrich (CA); compound C (CC) was purchased from APExBIO. Antibodies against LC3 (12741), phospho‐ACC (Ser79) (3661), phospho‐mTOR (Ser2448) (5536), phospho‐p70S6K (Thr389) (9206) and p53 (2524) were purchased from Cell Signalling Technology. Antibodies against p21 (ab109520) p53(ab31333), phospho‐AMPKa (Thr172) (ab133448), AMPKa1 (ab32047), β‐actin (ab8226) and SQSTM1/p62 (3340–1) were obtained from Abcam.

Lungs of the mice were analyzed using IHC with p53 rabbit polyclonal antibody (ab31333, Abcam), ACTA2 (α-SMA) rabbit polyclonal antibody (orb234994, Biorbyt) and ACACA (ACC1) rabbit polyclonal antibody (GTX132081, GeneTax). Data were analyzed with TissueFAXS (TissueGnostics, Vienna, Austria) based on the IHC staining slides.