Tcs sp5ii sted

After 4% paraformaldehyde fixation and 0.3% Triton X-100 permeabilization, the bovine serum albumin (BSA) was used to block cells, and rabbit anti-LC3B antibody (ab51520, 1:2000, Abcam) was later used to incubate cells under 4° C overnight, followed by another 1 h of incubation by the Alexa Fluor 488-labeled donkey anti-rabbit IgG secondary antibody (A21206, 1:500, Thermo Fisher Scientific, Waltham, MA, USA) under ambient temperature. Afterwards, we utilized the confocal laser microscope (Leica TCS SP5II STED, Mannheim, Germany) to observe cells. Altogether 200 or more cells were calculated from every section to determine the LC3 spot cell (autophagosome) proportion.

Cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS for 5 min each time, and then incubated with Triton X-100 (Sigma) for 15 min to increase membrane permeability. After cells were blocked with BSA for 1 h, they were left to incubate overnight with rabbit antibody anti-LC3B (ab51520, 1: 2000, Abcam) at 4 °C, and then with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (A21206, 1: 500, Thermo Fisher Scientific, USA) for 1 h in the dark at room temperature. Nuclei were left to stain with 4',6-diamidino-2-phenylindole in the dark for 15 min. Slides were dripped with anti-fluorescence attenuation sealing agent, and cells were observed under a confocal laser microscope (Leica TCS SP5II STED, Germany). At least 200 cells were counted in each slide and the percentage of LC3-positive cells (autophagosomes) was calculated.

Cells were fixed with paraformaldehyde (AWI0056B, Abiowell) for 30 min and incubated with 0.5% Triton X-100. The background was blocked BSA and cells were incubated with a primary antibody at 4 °C for 12 h followed by incubation with a fluorescent secondary antibody (AFIHC023; AiFang Biological) for 2 h at room temperature. After counterstaining with DAPI, the sections were observed under a confocal laser microscope (Leica TCS SP5II STED, Mannheim, Germany).

Exosomes were incubated with 5 μmol/L calcein-AM at 37 °C for 30 min. For in vitro experiments, cells were cultured in medium supplemented with labelled exosomes for 6 h, then fixed with 4% paraformaldehyde (PFA). Cells were then stained with Phalloidin (0.33 μmol/L) and DAPI (5 μg/mL) for 15 min. For in vivo experiments, labelled exosomes (100 μL) were injected into the adipose tissue from mouse hind limbs. Exosome internalization was allowed for 12 h, then mice were sacrificed. The adipose tissue was collected, fixed with 4% PFA, ethanol-dehydrated, embedded in paraffin, and sectioned. Tissue sections were stained with FABP4, a specific adipocyte marker, and DAPI and then visualized under a confocal laser microscope (Leica, TCS SP5II STED, USA).