The membranes were washed and reacted with peroxidase-conjugated anti-rabbit IgG antibody (Sigma Aldrich, MO, USA).
Following immunolabeling, membranes were washed and reacted with Super Signal chemiluminescence HRP substrate (Pierce Chemical Co).
After incubation for 18 h at 37°C, the culture medium was removed, the slides were washed, blocked, reacted with the mAbs and observed as described above.
Wells were incubated for 1 h at room temperature, washed, and reacted with a biotinylated anti-CD58 antibody (clone TS2/9.1.4.3), then, with HRP-conjugated streptavidin.
Sections were washed and reacted with secondary antibody conjugated either with Alexa 555, or Alexa 647, (Invitrogen, Eugene, OR) for 1 hour at room temperature before being washed and mounted on slides with Fluoromount-G (Southern Biotech, Birmingham, AL).
The sections were incubated with the EnVision plus Dual Link reagent (a polymer conjugated with goat anti-rabbit Ig or goat anti-mouse Ig and horseradish peroxidase) for 30 min. The sections were washed and reacted with 3-diaminobenzidine and hydrogen peroxide and counterstained with hemotoxylin for visualization by light microscopy.
Blots were then washed and reacted with electrochemiluminescence (ECL) or ECL-prime reagents.
Slides were then washed and reacted with a secondary antibody, anti-mouse Cy2 (1:300; Jackson Laboratories, ME) and anti-rabbit Cy5 (1:300; Jackson Laboratories, ME).
After activation, platelets were fixed with 1% paraformaldehyde, washed and reacted with a platelet-specific PE-labeled anti-CD41 mAb (clone 5B12; DAKO, Carpinteria, CA) and FITC-labeled ASKP1240 or FITC-labeled mu5C8.
After rehydrating, embryos were blocked again and incubated with HRP anti-DIG at 1 : 500 dilution for 4 h, then washed and reacted with TSA Plus Cy3 solution (Perkin-Elmer) for 45 min.
