Anti caveolin 1 antibody

Paraffin-embedded sections (4 μm thick) of subcutaneous AT were deparaffinized in xylene and rehydrated through descending grades of ethanol (100%, 95%, and 75%) to water. Antigen retrieval was performed by placing slides in a target retrieval solution (pH 6.0; Dako, Glostrup, Denmark) in a pressure cooker, boiling for 8 min, and cooling for 15 min. After washing in PBS, endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 min, and nonspecific antibody binding was blocked with 5% fat-free milk for 1 h, followed by 1% bovine serum albumin solution for 1 h. The slides were incubated at room temperature overnight with primary antibody (1:100 dilution of rabbit polyclonal anti-Caveolin-1 antibody; Cell Signaling Technology #3238s). After washing with PBS (0.5% Tween), slides were incubated for 1 h with a secondary antibody, namely, goat anti-rabbit conjugated with horseradish peroxidase (HRP) polymer chain Dako EnVision Kit from (Dako, Glostrup, Denmark) and color was developed using 3,3′-diaminobenzidine (DAB) chromogen substrate. Specimens were washed, counterstained, dehydrated, cleared, and mounted, as described elsewhere [25 (link)]. For analysis, digital photomicrographs of the entire AT sections (20X; PannoramicScan, 3DHistech, Hungary) were used to quantify the immunohistochemical staining using ImageJ software (NIH, Bethesda, MD, USA).

IHC staining was performed on formalin-fixed and paraffin-embedded sections of lung metastases with antibodies of anti-HMGCR (1:100, Santa cruz), anti-CCDC25 (1:100, Cell Signaling Technology), anti-MPO (1:200, R&D), anti-H3cit (1:300, Cell Signaling Technology), or anti-Caveolin-1 antibody (1:100, Cell Signaling Technology). Details can be found in the supplementary data.
Freshly obtained mouse lung tissues were quick-frozen in liquid nitrogen and placed in a cryostat to prepare frozen sections, each with a thickness of 4 mm. For IF staining, slides were rewarm at room temperature for 30 min and then combined with antibodies against CCDC25, Caveolin-1, MPO and H3cit overnight at 4 °C. Then rinsed the sections with ice PBS, and combined with secondary antibodies, respectively. Added DAPI-containing mounting medium, followed by mounting with a coverslip.

Cargo proteins were extracted from EVs by heating the vesicles at 60 °C for 15 min in 100 mM TrisHCl buffer containing 4% SDS. Proteins released from the ruptured vesicles were then mixed with gel loading buffer and boiled at 95 °C for 10 min. Protein samples were separated by SDS-PAGE on 12% polyacrylamide gels and then transferred onto nitrocellulose membranes at 100 V for 1 h. The membranes were probed with primary antibodies at 4 °C overnight. The antibodies used for Western blots were: anti-Alix antibody (#2171), anti-flotillin-1 antibody (#18634), anti-CD9 antibody (#13174), anti-caveolin-1 antibody (#3267S) from Cell Signaling Technologies (Danvers, MA, USA). The anti-hepatocyte growth factor receptor antibody (ab59884) and anti-FTH1 (ab75973) antibodies were from Abcam (Cambridge, UK). Anti-E-cadherin (sc-21791), anti-N-cadherin (sc-59987), anti-cathepsin B (sc-365558), anti-cathepsin D (sc-377299) and anti-IGFBP3 (sc-9028) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-PLOD2 (408105) antibody was obtained from Thermo Fisher Scientific. Protein–antibody conjugates were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific).