H1alpha67 chip grade

Retinal samples or samples of cells were sonicated in lysis buffer, namely mammalian protein extraction reagent (MPER; HyCell). Identical quantities of denatured proteins (40 μg/30 μl/well) then underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad) as described previously [10 (link)]. After separation, the protein bands were transferred to a polyvinylidene difluoride membrane, which was treated for 12 h at 4 °C with the following primary antibodies, mouse monoclonal anti-β-actin antibody (AC-15; 1:2000; ab6276)/anti-HIF-1α antibody (1:200; H1alpha67-ChIP Grade; Abcam Inc.), rabbit polyclonal anti-VEGF antibody (A-20; 1:200; sc-152)/anti-PKM2 antibody (1:500; ab38237), or rabbit monoclonal anti-RBP2 antibody (ab177486; 1:1000; Abcam Inc.). The blots were next treated with relevant secondary antibody, HRP-conjugated goat anti-rabbit IgG (1:5000; Santa Cruz Biotechnology Inc.) or goat anti-mouse IgG (1:5000; sc-2005) at 37 °C for 1 h. Dilution of primary/secondary antibodies was carried out in 5% fat-free skimmed milk. Finally, the membranes were processed using an enhanced chemiluminescent analysis system (HyCell) and exposed to an X-ray film (Fujifilm). The amount of each protein was then evaluated by scanning densitometry.

In IHC, paraffin blocks of the xenograft tumors were cut into 4-μm sections. The slides were incubated with primary antibodies: IGFBP3 (1:200, MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:500, H1alpha67-ChIP Grade; Abcam, Cambridge, UK), HIF-2α (1:1,000, ab8365; Abcam, Cambridge, UK), HO-1 (1:200, GTX101147, Genetex, Irvine, CA, USA), or Von Hippel-Lindau (VHL) (1:200, GTX101087, Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
In ICC, 0.2 × 10⁵ cells were cultured on 25 mm × 25 mm glass slides and incubated under hypoxic or normoxic conditions for 17 h. Then, the slides were incubated with primary antibodies: IGFBP3 (1:200; MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:300; GTX127309; Genetex, Irvine, CA, USA), or HIF-2α (1:300; GTX30114; Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
A chromogen in IHC and ICC was developed, following the protocol of UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, Waltham, MA, USA). The sections or slides were stained with hematoxylin for examination, and photos were taken using Moticam X3 Plus (Motic, Speed Fair Co., Ltd, HK) microscope camera by Motic Images Plus 3.0 software. The signals of IHC and ICC were quantified and analyzed by ImageJ 1.53 k software (NIH, USA).