Ab52917

Deparaffinized sections were subjected to immunohistochemistry as previously described. Antigens were retrieved on the slides using 10 mM sodium citrate and heated in a pressure cooker for 10 min. The slides were then immersed in H₂O₂ in methanol for 10 min to eliminate endogenous peroxidase activity. The sections were incubated with Goat serum for 10 min to reduce nonspecific reactions. They were then incubated with anti-CD31 pAbs (GB11063-2, Servicebio, Wuhan, China); antiSIRT1mAbs (ab110304, Abcam, USA), and anti-VEGFA mAbs (ab52917, Abcam) at 4 °C overnight. The slides were incubated with goat resistant mouse IgG polymer for 10 min. Then, they were incubated with HRP for 10 min. Color development was performed using a DAB substrate for approximately 5 min, and the specimens were counterstained with hematoxylin. The sections were examined under a microscope. SIRT1 and VEGF-A protein expression levels were quantified by blindly calculating the average optical density (AOD) in Image-Pro Plus (version 5.0). Each image was analyzed to obtain the integrated optical density (IOD) and area of the pixel (AREA) of a tissue. AOD was determined using the following equation: (AOD = IOD/AREA) [53 (link)]. Intrathrombotic CD31-positive microvascular density (MVD) was measured [34 (link)]. All measurements were performed by an examiner without prior knowledge of the experimental procedure.

The proteins were extracted by ice cracking. SDS-PAGE was used to separate the proteins. Subsequently, the target proteins were transferred onto a polyvinylidene difluoride membrane at low temperatures, followed by treatment with 5% milk for 50 min. Immediately afterward, the membranes were incubated overnight at 4°C with a primary antibody (ATAD2, 1 : 1000, #78568, Cell Signaling; VEGFA, 1 : 10000, ab52917, Abcam; and GAPDH, 1 : 5000, abs132004, Absin). In the following day, the membranes were removed and subjected to the process of PBST washing, treatment with a secondary antibody (anti-rabbit IgG, HRP-linked antibody, 1 : 5000, #7074 Cell Signaling), PBST rinsing, and, finally, exposure to the chromogenic apparatus by dropwise addition of the chromogenic solution for color development.

Femoral-head samples were fixed for 48 h in 4% paraformaldehyde and decalcified for 4 weeks in 10% ethylenediaminetetraacetic acid (G1105, EDTA, Servicebio, China). Then, the samples were embedded in paraffin and cut into 6 μm thick slices. H&E staining was performed to observe the general view of specimens and to evaluate the trabecular structure. For TRAP staining, paraffin sections were stained with a TRAP staining kit (G1050, Servicebio, China). Briefly, the paraffin sections were stained with tartrate buffer containing naphthol AS-BI phosphate and pararosaniline chloride at 37 °C for 1 h in darkness and counterstained with hematoxylin. Section images were acquired using an IX71-SIF microscope (Olympus, Japan). IHC staining was performed to define the expression of CST, as well as osteogenesis-, vascular-, and pathway-related markers. In brief, sections were dewaxed and gradient hydrated to retrieve antigens. Then, primary antibodies, including CST, osteocalcin (ab133612), VEGFA (ab52917), and CD31 (ab9498), and corresponding secondary antibodies (all from Abcam Cambridge, UK) were incubated. The chromogenic reaction was induced by a DAB Kit (Beyotime, China). The tissue sections were observed with an IX71-SIF microscope (Olympus, Japan). IHC-positive cells and areas were measured using ImageJ and counted by two independent observers.

After electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-rad, 1704159) and blocked for 1 h with 5% skimmed milk in TBST (0.1% Tween 20 in TBS). Primary antibodies [anti-VEGF-B (R&D systems, MAB3372) and anti-VEGF-A (Abcam, ab52917)] were incubated overnight at 4°C. After washes, membranes were incubated with secondary peroxidase coupled antibodies [anti-mouse (R&D systems, HAF018) and anti-rabbit (Invitrogen, 31460)] for 1 h at RT. Target proteins were detected using ECL western blot detection solution (Thermo Scientific, 32132).

A549, H226, HUVEC and HEK 293 T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs and A549 cells were cultured with F-12 K medium. H226 cells were cultured with 1640 medium. HEK 293 T cells were cultured with DMEM medium. All mediums were added fetal bovine serum to a final concentration of 10%. All cells were cultured with 5% (v/v) CO2 at 37 °C. For hypoxia, cells were cultured with 1% (v/v) O2 for 6 h. Antibodies for GSK3α (4337; 9338), β-actin (3700), HIF1β (5537; 3414), HIF1α-OH (3434), and HIF1α (36,169; 79,233) were purchased Cell Signaling Technology. Antibodies for VEGFA (ab52917), and flag (ab205606) were purchased from Abcam company.