Pab7888

Tissue microarray construction was conducted as previously described [36 (link)]. Two 1 mm diameter core biopsies were removed from the donor blocks. They were then transferred to defined array positions in the recipient paraffin block. Three tissue microarray (TMA) blocks (368 cases, Cohort 3) were constructed. We used the avidin-biotin-peroxidase complex method to perform the immunohistochemical staining. Briefly, rehydration and microwave antigen retrieval were performed first. Antibodies against human Sema3A (1:250, PAB7888; Abnova), F4/80 (1:250, ab100790; Abcam), CD163 (1:200, ab126756; Abcam) or CD68 (1:200, ab955; Abcam) were then applied to the slides, and incubated at 4°C overnight. The secondary antibody incubation (GK500705, Gene Tech, China) was then performed at 37°C for 30 min using 3,3′-diaminobenzidine and a Mayer's hematoxylin counter-stain.

Protein samples were extracted from exosomes, cells or tissues with procedures as described in detail elsewhere23 (link). Equal amounts of protein were subjected to SDS-PAGE. Binding of the primary antibody was detected by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Amersham Pharmacia), and bands were quantified with densitometry. The source of antibodies and dilutions used were as follows: rabbit anti-CD63 (sc-15363, 1:500 dilution), rabbit anti-CD81 (sc-9158, 1:400 dilution), and a primary antibody against Sema3A (#PAB7888, dilution 1:400, Abnova Co.), or Stat3 (SC-482, dilution 1:200 Santa Cruz Biotechnology). Either α–Actin or β-Actin (1:1000 dilution, Sigma-Aldrich) was used as an internal control.