Triple Immunofluorescent Staining of Telocytes

Triple immunofluorescent staining for CD34/Vimentin/PDGFRα was used as previously reported [17 (link), 18 (link)]. In brief, TCs were cultured on glass bottom cell culture dishes with 20 mm diameter glass (NEST, Nanjing, China) and were fixed in 4% paraformaldehyde containing 0.05% Triton-X-100 for 20 min. Then washed the dishes three times wash with 1 × PBS and blocked in 5% Bovine serum albumin (BSA) for 1 h. After incubated overnight at 4 °C with mouse anti-CD34 antibody, goat anti-vimentin antibody or rat anti- PDGFRα antibody (1:200 dilution; Abcam, Cambridge, UK) diluted in 1% bovine serum albumin (BSA) in PBS, the dishes were washing in PBS for three times. Then, dishes were incubated with APC conjugated anti-mouse secondary antibodies, PE conjugated anti-rat secondary antibodies and FITC conjugated anti-goat secondary antibodies (1:200 dilution; Jackson ImmunoResearch, USA). The nuclear were marked by DAPI according to the manufacture (KeyGEN BioTECH, Nanjing, China). Cells were observed and recorded under Olympus FV3000 Confocal Laser Scanning Microscope (DSS Imagetech Pvt. Ltd, New Delhi, India).