Ba0290

Eight glioma samples were fixed by immersion in 10% formalin solution and then embedded in paraffin. 10-µm thick tissue sections were deparaffinized and rehydrated (xylene × 2 for 10 minutes each, 100%, 95%, and 75% ethanol for 5 minutes each and deionized water for 5 minutes). The sections were incubated with 3% hydrogen peroxide in methanol for 10 minutes to quench peroxidase activity. Antigen retrieval was performed by boiling sections in 10 mM sodium citrate buffer (pH 6.0) for 10 minutes. After being rinsed with PBS, sections were blocked with normal goat serum for 20 minutes. The samples were incubated with primary anti-PD-L1 (1:200, ab237726, Abcam, Cambridge, UK) or anti-TGFβ antibody (1:200, BA0290, BOSTER, Wuhan, China) overnight at 4°C. The sections were then incubated with secondary antibody () for 30 minutes at room temperature. The staining was developed using 3,3’-diaminobenzidine (DAB) as substrate and counterstained with hematoxylin. The sections were developed using 3,3’-diaminobenzidine (DAB) as substrate and counter-stained with Mayer’s Hematoxylin. All sections were independently reviewed by two pathologists according to the WHO criteria.

Eight PDR proliferating membranes were obtained from six samples each in the intravitreal bevacizumab (IVB) treatment group and the untreated group. The samples were formalin-fixed, paraffin-embedded, cut into sections (5 μm thick), and then dewaxed and rehydrated. After the sections were washed with phosphate-buffered saline (PBS), they were incubated with streptavidin-peroxidase for 10 min, washed, and then blocked in goat serum. Sections were incubated with antibodies against the following antigens at 4° C overnight: transforming growth factor-β1 (TGF-β1, 1:250 dilution, BA0290, Bosterbio), CTGF (1:250 dilution, BA0752, Boster), alpha smooth muscle actin (α-SMA, 1:250 dilution, bs-10196R, Bioss), Collagen Type I Alpha 1 (COL1A1, 1:250 dilution, BA0325, Boster), fibronectin (FN, 1:250 dilution, BA1771, Boster), and vimentin (1:800 dilution, ab128507, Abcam). The sections were then washed with PBS and incubated with biotinylated goat anti-rabbit IgG (1:1000 dilution, ab150077, Abcam) for 1 h at room temperature. The samples were counterstained with DAPI and photographed using a fluorescence microscope. Fluorescence intensity was analyzed using image software and quantitative analysis was performed using ImageJ (Version 1.8.0, http://imagej.nih.gov/ij).

The immunohistochemical analysis was performed as previously described with some modifications[34 (link)]. Knee joint sections on slides were incubated with anti-TSP-1, TGF-β1, CTGF, VEGF and von Willebrand factor (vWF) antibody (BA2130, BA0290, BA0752, BA0407, BA0046, Boster, Wuhan, China). Subsequently, the sections were stained using polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and were visualized with DAB peroxidase substrate kit (ZLI-9017, ZSGB-BIO, Beijing, China). Rabbit anti-mouse vWF was used to identify endothelium as described before [41 (link)]. Following immunostaining, the synovium area in the joint of each section was evaluated under a microscope (LEICA DMi8) in three randomly selected areas at a magnification of 400×. The number of positively stained cells and total cells were counted by two different observers, and the means of the ratio of these two groups of cells were calculated. The number of microvessels positively stained with anti-vWF was also counted in the same area on the next serial section.