Gtx102643

Immunofluorescence staining was performed according to the manufacturer's instructions at 7 d post-UVB irradiation (n = 3). Eye paraffin sections were deparaffinized in dimethylbenzene and dehydrated in gradient ethyl alcohol as previously described [20 (link)]. Then, the sections were washed with PBS (phosphate-buffered saline) (0.1 mM, pH = 7.2 ± 0.1) 3 times for 5 min. Antigen retrieval solution (9 mL 0.1 mmol/L citric acid + 41 mL 0.1 mmol/L sodium citrate + 450 mL ddH₂0) was performed with a medium baking temperature for 10 min. Next, the sections were washed in PBS 3 times for 5 min. They were incubated with 10% goat serum for 2 hours, then the sections were incubated with anti-VEGF-α (GeneTex, GTX102643, USA) at 1 : 100 dilution at 4°C. The slides were washed with PBS and incubated with IgG (H+L) secondary antibody, Cy3 conjugate (Zhuangzhi, EK022, Xi'an, Shaanxi province, China) at 1 : 200 dilution for 1 hour. DAPI (100 ng/mL) was applied to stain the nuclear for 15 min. Images of slides were captured with a fluorescence microscope (BX53, Olympus, Japan).

For immunohistochemistry, uterine sections were dewaxed and rehydrated from graded ethanol, antigen retrieval by autoclave heating in citrate buffer (pH 6.0) (Diagnostic BioSystems; Cat#K035, Malvern, UK). Add enough hydrogen peroxide blocking solution (Abcam; Cat#ab64218, Cambridge, UK) to cover the sections. Incubate for 15 min at room temperature, the slides were incubated with rabbit polyclonal antibodies against vimentin (1:250; Abcam, Cat#ab92547, Cambridge, UK) or anti-cytokeratin 18 (1:500; Abcam, Cat#ab181597, Cambridge, UK) overnight at 4 °C, washed using Tris-buffered saline (TBS) containing 0.01% Tween-20 (TBS-T), and incubated with secondary antibody for 30 min at room temperature. The bound antibody was detected using the DAKO REALTM EnVisionTM Detection System Peroxidase/DAB+, Rabbit/Mouse reagent for 5 min. Subsequently, samples were incubated with anti-VEGF (1:50; GeneTex, Cat#GTX102643, Irvine, CA, USA) overnight at 4 °C, washed with TBS-T, and incubated with the biotinylated universal antibody for 30 min at room temperature, followed by R.T.U. Vectastain Elite ABC Reagent for 30 min at room temperature. The slides were counterstained with hematoxylin for 10 min. All slides were scanned using a Pannoramic SCAN (MIDI II, Sysmex Europe GmbH, Norderstedt, Germany), and a microscope was used to capture images.

Cells were homogenized in an ice-cold RIPA buffer (Keelton Co., Ltd., China). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo, Rockford, United States). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, United States) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBST) at room temperature for 2 h, incubated overnight with the primary anti-CDK6 (1:1,000 dilution, ab241554; Abcam), anti-VEGF (1:2,000, GTX102643; GeneTex), antiAT1R (1:2,000, ab124734; Abcam), or anti-GAPDH antibodies (1:2,000 dilution, #5174;CST). After washing with TBST three times for 10 min, the membranes were treated with HRP-labeled secondary antibodies (1:1,000 dilution, Beyotime, China) for 1 h and visualized using the chemiluminescence method (Thermo, Rockford, United States). The relative protein expression was measured using GAPDH as an internal control.