Goat antirabbit hrp igg

The exosomal fraction was lysed with 1× radioimmuno precipitation assay (RIPA; CMG, Iran) containing protease inhibitor (SIGMAFAST ™, USA) on ice. The protein concentration was determined using the Bradford method. Lysed exosomes were separated by electrophoresis in a 12% acrylamide sodium dodecyl sulfate (SDS) gel and then were transferred to NC membrane (BioRad, Hercules, and CA). The exosomal marker was visualized using primary antibodies (anti-CD63 antibody [rabbit IgG, System Biosciences, California] and anti-Histone H3 as a negative control [rabbit polyclonal, Bio-Legend, San Diego, CA]) and secondary antibody (goat antirabbit HRP IgG, System Biosciences, California). The blots stained with DAB as the chromogenic substrate in colorimetric detection.

Dot blot was used to detect exosomal marker (CD63, the transmembrane tetraspanin proteins) in the fraction containing lysed exosomes. The exosomal fractions were dropped into nitrocellulose (NC) membranes. The membranes were blocked with 5% nonfat dry milk and washed in Tris-buffered saline with 0.1% tween-20 buffer and probed with anti-CD63 antibodies (rabbit immunoglobulin G [IgG], System Biosciences, California). In the end, membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (goat antirabbit HRP IgG, System Biosciences, California). The blot was developed using 3,3’-diaminobenzidine (DAB) substrate.