Ab15011

Urinary EVs were heated for 5 min at 95˚aC with reducing Laemmli buffer and amounts corresponding to 1.5ml urine were loaded per each lane of a 10% SDS-PAGE gel. LNCaP cell lysate, prepared in 1x RIPA and heated with reducing Laemmli prior to loading, was used as a control (10 µg proteins per lane). PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (ThermoFisher Scientific) was loaded onto each gel for assessment of protein molecular weighs. After separation by SDS-PAGE, the proteins were transferred to Amersham Protran Supported 0.45 NC membranes (Merck Millipore), which were subsequently blocked using 10% (w/v) low-fat milk. Membranes were incubated with primary antibodies against TSG101 (Abcam, #ab15011, 1:1000 dilution), Calnexin (Abcam, #ab22595, 1:2000 dilution), or CD63 (Sino Biological, # 11271-T16, 1:300 dilution) overnight at + 4 ˚C. Membranes were washed in TBST and incubated for 1 h at room temperature with anti-rabbit IgG, F(ab’)2-HRP (Santa Cruz, #sc-3837, 1:2000 dilution), or goat anti-mouse m-IgG BP-HRP (Santa Cruz, # sc-516,102, 1:2000 dilution). After washing again in TBST, immunoreactive bands were visualized using Western Blotting Detection Reagent kit (GE HealthCare), and pictures were taken using a Nikon d610 dSLR body (Nikon) with Sigma 35 mm f/1.4 DG HSM Art lens (Sigma).

Tissue samples were decalcified in 10 mM ethylene diamine tetraacetic acid (EDTA) for 3 weeks, then dehydrated overnight with 15% and 30% sucrose, respectively, and finally embedded with optimal cutting temperature compound (OCT) (Sakura, Japan). Tissue sections of 10-μm thickness were prepared with a Leica cryostat. Cell samples were fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.3% Triton X-100 for 10 minutes and blocking with 3% BSA for 1 hour. Tissue sections or cells were incubated overnight with primary antibody at 4 °C. The primary antibodies used in this experiment are as follows: CD31(Abcam, ab182981, 1:200), CGRP (Abcam, ab36001, 1:200), Runx2 (Abcam, ab192256, 1:200), Sema3A (Abcam, ab23393, 1:200), Rock2 (Huabio, ER1706-48, 1:200), Drp1 (Abcam, ab184247, 1:200), and Mfn2 (Huabio, ER1802-23, 1:200). After washing the primary antibody, samples was incubated in the secondary antibodies for 2 hours. The secondary antibodies used are as follows: goat anti-rabbit Alexa Fluor 555 (Huanio, HA1123, 1:200), goat anti-rabbit Alexa Fluor 647 (Huabio, HA1123, 1:200), donkey anti-goat Alexa Fluor 555 (Abcam, ab15011). Immunofluorescence images were collected by a Nikon confocal microscope (Nikkon, N-Strom & A1, Japan).

The cultured embryonic lungs were immediately fixed in 4% formaldehyde, dehydrated and embedded in paraffin. Sections (5-6 μm) were prepared and deparaffinised. The sections were treated with 3% H₂O₂ and the antigen retrieval was achieved in 10 mM citrate buffer (PH 6.0). Immunohistochemistry of surfactant proteins SP-C and SP-B was performed using primary antibody Goat anti-SftpC (1:200, Santa Cruz, (C-19): sc-7705) and Rabbit anti-Pro SftpB (1:600, Abcam, ab15011). A Streptavidin-HRP conjugated Rabbit anti-Goat IgG anti-HRP (1:200, Bethyl, A50-100p) and Goat anti-Rabbit HRP (1:100, Abcam, ab97051) secondary antibody and the DAB Substrate Kit (DAB Substrate Kit, Cell Signaling, #8059) was used for detection. The nuclear counter stain was performed using haematoxylin.