Donkey anti rabbit immunoglobulin

For in vitro experiments, olaparib (Selleckchem, Planegg, Germany) was used at 5 μm for 24 h. For in vivo experiments, olaparib (MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in DMSO and further diluted in 10% (2‐hydroxypropyl)‐β‐cyclodextrin (Sigma Aldrich, Darmstadt, Germany) in PBS. olaparib was administered intraperitoneally at a dose of 50 mg/kg. Treatment with olaparib or vehicle took place three times a week for 3 weeks.
Antibodies and dilutions for tissue immunofluorescence staining were rat CD34 (BD553731; BD Biosciences, Franklin Lakes, NJ, USA), 1:25; rabbit VE‐cadherin (ab205336; Abcam, Cambridge, UK), 1:500; mouse S100B (S2532; Sigma Aldrich), 1:100; mouse SMA‐Cy3 (C6198; Sigma Aldrich), 1:200; donkey anti‐rat immunoglobulin (Jackson Immunoresearch, Ely, UK), 1:200; donkey anti‐rabbit immunoglobulin (Jackson Immunoresearch), 1:200; and horse anti‐mouse immunoglobulin (Vector Laboratories, Burlingame, CA, USA), 1:100.

Total proteins from cells, tissues, and exosome samples were extracted using a radioimmunoprecipitation assay (RIPA) kit (Beyotime Biotechnology, Jiangsu, China). They were separated on polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-SCAI (ab124688; Abcam, Cambridge, UK) antibodies at 4°C overnight and were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG at room temperature for 1 h. To identify exosome markers, we purchased primary antibodies against TSG101 from Abcam (ab125011; Cambridge, UK) and obtained primary antibodies against Hsp70 from Cell Signaling Technology (#4873; CST, Beverly, MA, USA). The secondary antibodies were F(ab)₂ fragments of donkey anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin linked to horseradish peroxidase (Jackson ImmunoResearch, USA). Immunoblotting reagents from an electrochemiluminescence kit were used (Amersham Biosciences, Uppsala, Sweden).

To identify exosome markers, primary antibodies against CD63 and TSG101 were purchased from Abcam (Cambridge, UK), and primary antibodies against Hsp 70 and Hsp 90 were obtained from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, USA). Immunoblotting reagents from an electrochemiluminescence kit were used (Amersham Biosciences, Uppsala, Sweden).