Anti fibrinogen antibody

Mice were primed intradermally with 5 μg lipopolysaccharide (LPS) (Sigma) or normal saline control (no‐priming) in the foot and challenged 24 h later by intravenous injection of 300 μg LPS. Blood and tissue samples were obtained 6 h after LPS challenge. Each group was composed of 3 mice. Anesthetized mice were perfused with 20 mL of PBS through the left ventricle. The liver was removed, fixed in 10% formalin, and embedded in paraffin. After rehydration, antigen retrieval, peroxidase blocking, and protein blocking, sections were incubated overnight with anti‐fibrinogen antibody (Abcam) in a humidified chamber. Goat anti‐rabbit IgG peroxidase (Servicebio, Wuhan, China) was used as the secondary antibody for 30 min. Antigen‐antibody complexes were visualized with DAB Peroxidase Substrate.

Biopsy and immunohistochemical staining was conducted as described previously^{15 (link)}. Briefly, liver biopsy samples from the recipient monkeys were fixed in 10% neutral buffered formalin, and embedded in paraffin. Paraffin-embedded tissues were 4-μm sectioned using a microtome. Each de-paraffinized and hydrated section was incubated for 30 min with primary antibody cocktails for insulin (Santa Cruz Biotechnologies, Dallas, TX), CD3 (DAKO, Agilent, Santa Clara, CA), CD4 (Santa cruz Biotechnologies), CD8, CD20, and CD68 (all from Abcam, UK), and then washed four times in TBST. After staining procedure, all of the stained slides were dried at 60 °C, and mounted with aqueous mounting medium (Thermo Fisher Scientific). For immunofluorescence staining, anti-fibrinogen antibody and anti-insulin antibody (both from Abcam) were used as primary antibodies and, Alexa 488-conjugated anti-rabbit IgG and Alexa 647-conjugated anti-mouse IgG were used as secondary antibodies, respectively. FITC-conjugated anti-monkey IgG (Acris, Germany) was used to detect antibody involvement at the graft site. The stained sample was observed by Carl Zeiss Axio Imager A1 microscope and images were taken with a micrograph with AxioVision software (Carl Zeiss, Germany).