Mouse on mouse biotinylated anti mouse igg reagent

Paraffin sections (4 µm thick) were subjected to antigen retrieval (10 mmol/L citrate buffer, pH 6.0) for 25 minutes at 95°C. Endogenous peroxidase and endogenous biotin were blocked using hydrogen peroxide (3%) and avidin/biotin blocking kit (Vector Laboratories), respectively. Sections were incubated with mouse IgG per the mouse-on-mouse biotinylated anti-Mouse IgG reagent (Vector Laboratories), and staining for fibrin was performed using mouse anti-human fibrinogen antibody (dilution 1:1000 ; gift from Dr. C Esmon, Oklahoma Medical Research Foundation) [30 (link)]. Subsequently, sections were incubated with the mouse-on-mouse biotinylated anti-Mouse IgG reagent, followed by Vectastain ABC kit reagents (Vector Laboratories). Slides were developed using ImmPACT DAB Peroxidase substrate (Vector Laboratories) and counterstained with hematoxylin (Dako). Fibrin staining was graded in a blinded fashion for fibrin deposition by using a scale from 0 (no deposition) to 4 (abundant fibrin signal).