Goat anti rabbit igg h l

Immunostaining was performed on a Dako Autostainer per manufacturer’s instructions (CSA kit and Genpoint kit, Agilent) [38 , 40 ]. Each slide was incubated with a single primary antibody at room temperature for 30 min (Supplemental Table 2), and antibody specificity was confirmed by Western blotting as previously described [41 ]. The negative control slide was incubated with antibody diluent. Secondary antibody was goat anti-rabbit IgG H + L (1:10,000) (Vector Labs) or anti-mouse IgG (1:10, Agilent Dako CSA kit).
Spot (pixel) intensity with adjusted background correction was analyzed using ImageQuant v5.2 (Molecular Dynamic) [36 , 39 ]. Data reduction algorithm (RASv16, http://capmm.gmu.edu/rpma-analysis-suite) was used to quantify and normalize the relative intensity value to the total protein/spot [38 ].

The slides for the lung and the brain were deparaffinized with xylene and dehydrated through a gradient (100%, 90%, 80%, and 70%) ethanol series. The slides were immersed in 3% H₂O₂ (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes at room temperature to inhibit endogenous peroxidase activity and incubated with 10% normal goat serum for 1 hour at room temperature. The slides were washed three times in PBS for 5 minutes and incubated with CD63 and CD81 antibodies in 1× PBS with 5% normal goat serum for 1 hour at room temperature. After the incubation, the slides were washed three times with PBS for 5 minutes and incubated with biotinylated secondary antibody, goat anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA, USA) for 1 hour at room temperature. The slides were washed three times with PBS for 5 minutes and incubated with avidin-biotin-peroxidase complex solution (Vector Laboratories) for 1 hour at room temperature. The slides were washed three times with PBS for 5 minutes and treated with DAB solution [DAB (3, 3-diaminobenzidine) peroxidase (HRP) substrate kit (with nickel)] (Vector Laboratories) for 2 minutes. The slides were immersed in 70%, 80%, 90%, and 100% ethanol, as well as xylene, successively. The expression of the exosome markers CD63 and CD81 on the lung and the brain was detected by ImageJ software.