Bs 0778r

Cells were fixed in 4% paraformaldehyde for 30 min followed by washing for three times using PBS. Cells were treated with 0.1% Triton X-100 for 15 min and blocked using 5% BSA (Hyclone, SH30574.03) for 1 h. For renal tissue, specimen was fixed in 4% paraformaldehyde for 24 h or more. Through the production of paraffin sections and antigen retrieval, tissue sections were blocked using 5% BSA for 1 h. Then, cells and sections were incubated using primary against CD29 (sc-9970, 1:200, Santa Cruz), CD34 (bs-0646R, 1:500, bioss), CD44 (bs-4916R, 1:400, bioss), CD45 (bs-0522R, 1:300, bioss), CD90 (bs-0778R, 1:500, bioss), nephrin (sc-377246, 1:100, Santa Cruz), LC3 (12135-1-AP, 1:200, Proteintech) at 4 °C overnight. After incubation with secondary antibodies at 37 °C for 1 h, samples or cells were stained with DAPI for 5 min and laser confocal microscopy was adopted for observation and photo taking.

In this study, we used promyelocytic leukemia zinc finger (PLZF) and Thy-1 cell surface antigen (THY1) as specific markers of goat SSCs for immunofluorescence staining. Briefly, the cells were pipetted into a 96-well plate (3599; Corning) precoated with poly-L-lysine (P2100; Solarbio), with approximately 5 × 10³ cells per well. Then, the cells were fixed in 4% paraformaldehyde (PFA; P1110; Solarbio) for 15 min and then permeabilized by the addition of 0.25% Triton X-100 (T8200; Solarbio). Thereafter, 10% of goat serum was added for blocking. A diluted primary antibody (1 : 200; bs-5971R and bs-0778R; Bioss, Beijing, China) was added and incubated overnight at 4°C. After rinsing with PBST, a diluted fluorescent secondary antibody (1 : 200; bs-0295G-Cy3 and bs-0295G-FITC; Bioss) was added and incubated for 1 h at room temperature. After incubation, the cells were rinsed three times with PBST. We added 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; C0060; Solarbio), and the cells were rinsed after 5 min of incubation. The cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan).