Ab182981

Heart samples were harvested, fixed with paraformaldehyde, and embedded in paraffin. Hematoxylin and eosin (HE) and Masson’s trichrome staining were performed. To quantify the fibrosis area, ten fields of view were randomly selected from eight cardiac sections and the percentage of Masson’s trichrome positive-stained area relative to total myocardial area was calculated. TUNEL staining was performed using a TUNEL apoptosis detection kit (40306ES50, Yeasen, China), according to the manufacturer’s instructions. Immunohistochemical staining was performed according to the standard protocol and using the following antibodies: Collagen type 1 (Col I, 1:1,000; ab34710, Abcam), αSMA (1:500; #19245, CST), CD31 (1:1,000; ab182981, Abcam), CD206 (1 µg/mL; ab64693, Abcam), goat anti-rabbit IgG H&L (HRP) (1:10,000; ab205718, Abcam), and goat anti-mouse IgG H&L (HRP) (1:10,000; ab205719, Abcam). For immunofluorescence, cells were fixed in cold methanol for 5 min, then permeabilized and blocked in phosphate buffered solution (0.5% Triton X-100) with 1% bovine serum albumin for 1 h. Immunostaining was performed using the following antibodies: Collagen type 1 (ab34710, Abcam), αSMA (#19245, CST), and the corresponding secondary antibodies.

For hematoxylin and eosin (H&E) staining, the mouse lung tissues were fixed with 4% paraformaldehyde for 24 h. The paraffin-embedded tissues were cut in 5 µm thick sections. H&E staining was employed to assess the alterations in morphology in these tissues using an optical microscope, and different fields were subsequently photographed.
For immunohistochemistry (IHC) analysis, paraffin-embedded tumor and lung tissues were deparaffinized and rehydrated as described [17 (link)]. After antigen retrieval and blocking, sections were incubated with anti-CD31 (1:50, ab182981, Abcam, Cambridge, UK), anti-TIMP2 (1:200, ab180630, Abcam) or anti-TIMP3 (1:100, ab213063, Abcam) antibody at 4 °C overnight, followed by the incubation with secondary antibody. The immunoreactivity was visualized by using DAB substrate (Thermo Fisher Scientific).

The osteosarcoma tissue was embedded in paraffin and cut into 5-μm-thick sections. The sections were then dried overnight at 37 °C, dewaxed in xylene, and rehydrated with graded alcohol. Next, the tissue sections were quenched in 3% hydrogen peroxide, blocked with 5% BSA for 1 h, and immunostained with primary antibody against CD31 (ab182981, 1:2000, Abcam) at 4 °C overnight. After PBS washing, the sections were incubated with the secondary antibody for 1 h. All sections were incubated with Envision system-HRP substrate using a Dako REAL Ebision kit (DAKO, Glostrup, Denmark) before observation under a microscope (Leica). Evaluation of MVD in tumor tissues was implemented referring to a previous study [14 (link)].

Brain tissue slides were blocked in 1% bovine serum albumin/phosphate-buffered saline for 1 h at RT and incubated with anticluster of differentiation 31 (CD31) (1 : 200, Cat. No. ab182981, Abcam), anti-ionized calcium binding adaptor molecule 1 (1 : 250, Cat. No. ab178847, Abcam), antiglial fibrillary acidic protein (1 : 400, Cat. No. ab49874, Abcam), and antineuronal nuclear protein (1 : 250, Cat. No. ab177487, Abcam) at 4°C for overnight, followed by incubation with corresponding secondary antibodies at RT for 1 h. The experiment was repeated three times with consecutive brain slices from each group, and the slides were observed by fluorescence microscopy in a blinded fashion.

Lung tissue sections were dewaxed, rehydrated, and then washed with PBS. After antigen retrieval, the sections were stained for anti-α-smooth muscle actin (α-SMA, Abcam, #ab7817) antibody and antiproliferating cell nuclear antigen (PCNA, Cell Signaling Technology, #2586) antibody or anti-α-SMA antibody and antiplatelet endothelial cell adhesion molecule-1 antibody (PECAM-1/CD31, Abcam, #ab182981) with appropriate fluorescent secondary antibodies and DAPI mounting medium. All slides were scanned using an AperioScanScope (Leica), and representative images were taken at ×100 by an individual blinded to experimental condition.

We performed immunofluorescence staining on brain tissue sections and HBMECs. The tissue sections were dewaxed with xylene and then rehydrated in ethanol. High glucose‐treated HBMECs were fixed on cell slides with 4% paraformaldehyde. The fixed sections and cell slides were transferred to blocking solution (goat serum) for 1 h. The blocked HBMECs were incubated overnight at 4°C with primary antibodies against SREBP1 (AF6283, diluted 1:100, Affinity Biosciences) and p‐mTOR (cst5536, diluted 1:100, Cell Signaling Technology). The tissue sections were incubated overnight at 4°C with primary antibodies against SREBP1 (AF6283, diluted 1:200, Affinity Biosciences), LRP1 (ab92544, diluted 1:500, Abcam), Raptor (DF7527, diluted 1:200, Affinity Biosciences) and CD31 (ab182981, diluted 1:2000, Abcam). Immunoreactions were observed using CY3‐conjugated affinity‐pure goat anti‐rabbit IgG (BA1032, diluted 1:100, Bost Biotech) and FITC‐conjugated affinity‐pure goat anti‐mouse IgG (BA1011, diluted 1:100, Bost Biotech). Nuclei were stained with Hoechst (Invitrogen). Antifluorescent quenchers were added, and the slides were observed under a microscope (Olympus CX31 microscope).