Rabbit anti f4 80

Liver segments of piglets were subjected to immunofluorescence staining. Briefly, six micron-thick slices were obtained from paraffin-embedded livers. Xylene and a series of alcohols were used for section dewaxing. After being soaked in citrate antigen retrieval solution (Beyotime Biotechnology, P0081, Shanghai, China), 0.3% Triton X-100 (Beyotime Biotechnology, ST797), and 3% H2O2-methanol, slices were blocked with 5% BSA for 1–2 h at room temperature. Next, specimens were incubated at 4 °C overnight with rabbit-anti-F4/80 (Servicebio, GB113373, Gent, Belgium) and cultured for 1 h with the secondary antibody, Alexa Fluor Plus 647 goat anti-rabbit IgG (Invitrogen, A32733). After all antigens had been labeled, nuclei were stained with Hoechst 33342 (Invitrogen, H3570). Images were acquired using a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) and analyzed by ImageJ program V1.8.0.112 (National Institutes of Health, Bethesda, MD, USA).

The liver tissues were fixed in 4% paraformaldehyde for over 24 h, paraffin-embedded, cut to 4 μm thick, and stained with hematoxylin and eosin (H&E), Sirius red (SR) and immunohistochemical (IHC) staining. The IHC staining was used to analyze epithelial cell proliferation (cytokeratin 19, CK19), immune cell infiltration (mouse EGF-like module-containing mucin-like hormone receptor-like 1, EMR1, F4/80), and liver fibrosis (α-smooth muscle actin, αSMA) in the bile duct. We performed IHC staining with the following primary antibodies: (a) rabbit anti-αSMA (Servicebio, Wuhan, China, GB111364); (b) rabbit anti-CK19 (Servicebio, GB11197); and (c) rabbit anti-F4/80 (Servicebio, GB113373). Standard procedures of IHC staining were used as described in a previous study [9 (link)]. Representative images were randomly selected and recorded by specialized pathologists without knowledge of the group assignment in a blinded manner using Panoramic scanning (3DHISTECH, Budapest, Hungary). The images were then analyzed and quantified using ImageJ.