Ab92498

Tissue samples (basal cortical samples) were pooled and cell samples were lysed and resolved using 12% SDS-PAGE. Subsequently, the resolved proteins were blotted onto a membrane, which was then blocked at room temperature for 1 h with Tris-Buffered Saline and Tween 20 (TBST) containing 5% bovine serum albumin (BSA) and incubated at 4°C overnight with primary antibodies against HIF1A (ab92498, Abcam, Cambridge, MA) and TLR4 (ab8378, Abcam, Cambridge, MA). After TBST washing, the membrane was further incubated for 1 h at 37°C with HRP-labeled second antibodies (ab6728, Abcam, Cambridge, MA). Finally, after being developed using chemiluminescence reagents (Santa Cruz Biotech, Santa Cruz, CA), the optical density values of target proteins were measured.

Cells, 4 × 10⁴, were plated on glass slides and cultured overnight. Then cells were fixed with 4% paraformaldehyde (15 min) and permeabilized with 0.5% Triton-100 for 10 min. Four percent BSA was used to block non-specific protein–protein interactions (1 h). The cells were treated with antibody against human HIF1-a (ab92498, Abcam) overnight followed by anti-rabbit Alexa Fluor 488 (ab150077, Abcam) and Alexa Fluor 594 (R37117, Thermo Fisher) secondary antibodies. After counterstaining with DAPI, cells were recorded, and the mean optical density was researched by Image J.

Histochemical immunostaining assay was done using a streptavidin-peroxidase (SP) three-step method. All samples were first fixed in 4% formalin and embedded in paraffin. After antigen repair, the slides were incubated overnight at 4°C with primary antibodies against HIF1A (ab92498, Abcam, Cambridge, MA) and TLR4 (ab8378, Abcam, Cambridge, MA). After being washed repeatedly with PBS, the slides were incubated for 10 min at room temperature with the secondary antibody. Subsequently, the reaction was terminated by an anti-biotin-labeled peroxidase solution, and the slides were colorized with DAB. After re-staining with hematoxylin, the slides were dehydrated with anhydrous ethanol and dried before they were mounted in neutral gum and observed under a microscope.

The total proteins were extracted from HPAEpiCs or lung tissues by using lysis buffer (P0013G, Beyotime, China), and the protein concentration in each extract was determined using a Bradford protein assay kit (ab102535, Abcam, Cambridge, UK. Next, a 20 ug sample of total protein from each extract was separated by SDS-PAGE (10%; Beyotime), and the protein bands were transferred onto PVDF membranes (Millipore, Billerica, MA, USA), which were subsequently incubated overnight at 4°C with the following antibodies: anti-LC3B (1 : 1000, ab51520, Abcam, Cambridge, MA, USA), P62 (1 : 1000, ab91526, Abcam), Beclin-1 (1 : 1000, ab207612, Abcam), SLUG (1 : 1000, ab51772, Abcam), HIF-1α (1 : 500, ab92498, Abcam), E-cadherin (1 : 500, ab40772, Abcam), N-cadherin (1 : 1000, ab202030, Abcam), and GAPDH (1 : 10000, ab8245, Abcam). Next, the membranes were incubated with an HRP goat anti-rabbit/mouse IgG secondary antibody (1 : 20000, ab8245, Abcam); after which, the immunostained protein bands were detected using an enhanced chemiluminescence detection system (Millipore). The integral optical density values of the protein bands were analyzed using Image Pro Plus 6.0 software.

Protein extraction from HESCs or endometriotic lesions was carried out using RIPA lysis buffer (Solarbio), and a bicinchoninic acid assay kit (Solarbio) was utilized for measuring the protein concentration. Protein samples (20 μg) were dissolved with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto polyvinylidene fluoride membranes (Beyotime), and blocked with 5% defatted milk. Next, the membranes were incubated overnight with anti-HIF1AN (ab92498, 1:5,000), anti-HIF1A (ab179483, 1:1,000), anti-VEGF (ab214424, 1:1,000), and anti-GAPDH (ab22555, 1:1,000) primary antibodies (all from Abcam) at 4℃, and then incubated at room temperature with the secondary antibody (ab6721, 1:2,000, Abcam) for 2 h. Protein bands were visualized with the enhanced chemiluminescence detection kit (Solarbio) and quantified with ImageJ software (NIH).

Tissue sections were prepared by Drs. C. David James and Jann Sarkaria's group at Mayo Clinic [46] (link), [50] . An immunohistochemistry accessory kit (Bethyl laboratories, Inc., Montgomery, TX) was used to examine expression levels of FIH-1 according to the manufacturer's protocol. Briefly, after deparaffinization and rehydration, the slides were incubated in 3% hydrogen peroxide/methanol to quench endogenous peroxidase. Slides were then incubated in hot Epitope retrieval buffer (provided in kit) to recover epitopes for 20 min at 90–96°C. Non-specific reactions were blocked by incubating the sections with blocking reagent (included in the kit) for 30 min at room temperature, then 200 µl of human FIH-1 antibody (dilution 1∶200,catalog ab92498, Abcam, Cambridge, MA) was applied to each slide and incubated overnight at 4°C. Slides were then incubated with working anti-rabbit IHC antibody (provided in kit), and peroxidase activity was visualized with working 3, 3′-diaminobenzidine (DAB) substrate (provided in kit). Counterstaining was performed with hematoxylin. The stained sections were randomly selected high-power fields (×40).