Igg h l alexa fluor 647

The expression of NRP1 in human patients and mouse tumour tissues were examined by immunohistochemistry. The samples were fixed with 4% paraformaldehyde at room temperature overnight, dehydrated and embedded in paraffin, and cut into 5-mm thick sections. The sections were incubated at 60°C for 4 h, dewaxed using xylene and rehydrated using a decreasing ethanol gradient (100, 95, 75 and 50%, 5 min each time). After washed three times for 5 min with PBS, sections were incubated in 3% hydrogen peroxide at room temperature for 10 min to inactivate endogenous peroxidase. Sections were heated at 95°C for 20 min in EDTA bufer and natural fall to room warm. The sections were blocked in 5% BSA (B2064; Sigma-Aldrich; Merck KGaA) and 0.3% Triton X-100 (T8200; Solarbio) for 30 min. Tissues were incubated with rabbit monoclonal human primary antibody (1:200; cat. no. ab81321; Abcam) overnight at 4°C and goat anti-rabbit IgG H&L (Alexa Fluor^® 647) antibody (1:200; cat. no. ab150079; Abcam) for 2 h at room temperature in dark. The sections were stained in 50 µl DAPI solution and incubated in dark for 10 min at room temperature. For each slice, images of five sections were acquired under using a light microscopy and analysed using cellSens Standard 1.18 (Olympus Corporation).

ApoVs were permeabilizated (Biolegend,
USA) for 3–5 min and rinsed five times in 0.1 μm-filtered
PBS at 16 000g for 30 min. Next, apoVs were
incubated in the first antibody (GOLPH2, 1:50, Abcam, UK) overnight
at 4 °C and rinsed two times. After apoVs were stained with IgG
H&L (Alexa Fluor 647, Abcam, UK) for 1–2 h and rinsed two
times, MSCs were incubated with those apoVs for 24 h. The remaining
steps refer to CLSM.