Rabbit anti hsp70

The proteins from UFs‘ EVs and the endometrium were extracted by RIPA buffer (Beyotime, Cat. No. P0013B) supplemented with 1% PMSF (Beyotime, Cat. No. ST506). The sample was denatured by heating, separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Cat. No. IPVH08110), washed four times (5 min each time) with TBST, and blocked with 5% skim milk in TBST for 2 h at room temperature. The following antibodies were incubated with the membranes after being washed four times with TBST: rabbit anti TSG101 (ZEN-BIOSCIENCE, Cat. No. abs127362, 1:1000 in TBST), rabbit anti HSP70 (Proteintech, Cat. No. 25682-1-AP, 1:1000 in TBST), and rabbit anti Calnexin (Proteintech, Cat. No. 10427-2-AP, 1:1000 in TBST) overnight at 4 °C. After being washed four times with TBST, the membranes were incubated with HRP-labeled Goat Anti-Rabbit secondary antibodies (Beyotime, Cat. No. A0208, 1:1000 in TBST) for 2 h at room temperature. The images of the membranes were captured by a UVP system (Upland) after they were treated with an enhanced chemiluminescence (ECL, Beyotime, Cat. No. P0018S) reagent.

Discarded human tumor skin tissues were processed at the Department of Pathology at Stanford University and sectioned at a thickness of 5 μm. Immunostaining was performed on paraffin sections. Heat-based antigen retrieval was performed when necessary. Tissue sections were blocked in either 3% bovine serum albumin (BSA) or 3% donkey serum. The following primary antibodies were used: rabbit anti-CLIC2 (Abcam; 1:50), rabbit anti-CEMIP (Proteintech; 1:100), rabbit anti-TMEM119 (Proteintech; 1:50), chicken anti-KRT14 (BioLegend; 1:1000), mouse anti-LHX2 (Santa Cruz Biotechnology; 1:50), rabbit anti-ACTA2 (Abcam; 1:250), mouse anti-KRT15 (Santa Cruz Biotechnology; 1:50), rabbit anti-HSP70 (Proteintech; 1:100), rabbit anti-CASP3 (R&D Systems; 1:1000), and rabbit anti-MKI67 (Abcam; 1:1000). Secondary chicken (Abcam), rabbit (Life Technologies), and mouse (Life Technologies) were used at a concentration of 1:1000. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Images were acquired on an Olympus FV3000 confocal laser scanning microscope.