Rabbit anti hif 1α d2u3t

Nuclear proteins were extracted from hypoxic and normoxic cells using a NE-PER nuclear and cytoplasmic extraction kit (Cat# 78833, Thermo-Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Equal amounts of nuclear protein were electrophoresed in 10% SDS-polyacrylamide gels and transferred to hybond-PVDF membranes (Cat# 23225, GE Healthcare Life Sciences, Pittsburgh, PA, USA). The primary antibodies used in this study were: rabbit anti-Lamin B1 (Cat# ab133741, Abcam, Cambridge, MA, USA) and rabbit anti-HIF-1α (D2U3T) (Cat# 14179, Cell Signaling Technology, Danvers, MA, USA). Next, the membranes were incubated with anti-rabbit Ig, peroxidase-linked, species-specific whole antibody (Cat# 31460, Thermo-Fisher Scientific). ECL reagents were used to detect the signals according to the manufacturer’s instructions (Cat# RPN3244, GE Healthcare Life Sciences, Pittsburgh, PA, USA). All films were scanned with an Epson Expression 1680 optical scanner (Epson America Inc., Long Beach, CA, USA). Densitometry analysis of bands was performed using an open-source, public domain software package (ImageJ v1.47).

To visualize cellular morphology, A375 tumor sections were stained with hematoxylin and eosin (H&E). Gram staining of A375 tumor specimens was performed using a Gram stain kit (#AR17592-2, Agilent Technologies, Santa Clara, CA, USA) following the standard protocol for paraffin specimens and according to the manufacturer’s instructions. For immunohistochemistry, tumors were excised, fixed in 10% formalin, embedded in paraffin blocks, and processed for histological analysis. Rabbit anti-HIF-1α (D2U3T) (Cat# 14179, Cell Signaling Technology, Danvers, MA, USA) was used at a 1:500 concentration to detect expression of HIF-1α. The slides were then washed with PBS 1×, incubated with the standard ultra-sensitive ABC peroxidase staining kit (Pierce, Rockford, IL, USA), and detected with diaminobenzidine (DAB) tetrahydrochloride solution containing 0.0006% H₂O₂. Hematoxylin was used as a counterstain. Whole tissue slides were scanned with Leica Aperio ImageScope with 40× magnification (Leica Biosystems, Buffalo Grove, IL, USA).