Anti human fibrin ogen antibody

Immunocytochemistry was performed using standard techniques. In lung and liver tissue, anti GP9 antibody (bioorbyt, #orb 167288) was used to detect platelets, neutrophils were detected with Ly6G antibody (BD, #551459). Primary antibodies were visualized with goat anti rabbit Alexa Fluor 568 and goat anti rat Alexa Fluor 568 secondary antibodies (Invitrogen). For platelet and neutrophil localization, an Axio Observer.D1 (Carl Zeiss) was used. To determine active caspase-3, anti caspase 3 antibody (Cell Signaling, #9661,) was used and visualized with the secondary antibody donkey anti rabbit Alexa Fluor 555 (Thermo Fisher, #31572) to detect cell apoptosis in lung and liver tissue. Fibrin(ogen) staining was performed using anti human fibrin(ogen) antibody (Dako, #2022-03) that was visualized with the secondary antibody goat anti rabbit Alexa fluor 488 (Invitrogen).

Brains for immunohistochemistry were cut axially at a thickness of 40 μm. Free floating sections were washed 3 times in PBS + 0.3% Triton X100, pH 7.4 prior to incubation in blocking solution (PBS, 3% BSA, 0.3% Triton X100, 3% donkey serum) for 3 hours at room temperature.
Primary and secondary antibodies were incubated in blocking solution overnight at 4°C. Fibrinogen was stained using the polyclonal anti-human fibrinogen antibody (Dako, A0080, 1:100). Erythrocytes were stained using the rat anti-mouse Ter119 antibody (BD Pharmingen, 553671, 1:100). Mouse IgG was stained and detected using the donkey anti-mouse IgG-Cy5 antibody (Jackson ImmunoResearch, 715-175-150, 1:200). Staining for fibrinogen and erythrocytes was detected using donkey anti-rabbit-Cy3 antibody (Jackson ImmunoResearch, 711-162-152, 1:500) and donkey anti-rat 488 (Jackson ImmunoResearch, 712-545-150, 1:500), respectively. Whole sections were imaged on the TissueScope 4000 fluorescence tissue scanner (Huron Digital Pathology) at a scan resolution of 0.5 μm/pixel and constant PMT settings.