Anti mouse igg h l alexafluor488

The HCC tissue was snap‐frozen in optimal cutting temperature compound and stored at −80°C for several days before cryosectioning into 20‐μm thick slices. Sections were thawed at room temperature. Then the tissue slices were fixed with 4% PFA for 15 min and then permeabilised with 0.5% Triton‐X in PBS for 30 min. Samples were blocked with blocking buffer containing 5% BSA and 0.1% Triton‐X in 1× PBS for 30 min, incubated with primary antibodies and secondary antibodies at room temperature for 1 h, respectively. Antibodies were diluted in blocking buffer. The antibody was removed by washing the samples three times in 1× PBS at room temperature for 5 min each time. Finally, the samples were post‐fixed in 4% PFA for 10 min and then we performed DNA FISH as previously described. For HCC tissue slices immuno‐FISH, rabbit anti‐PECAM1 (Abcam; Ab28364; 1:50) (TEC marker), rabbit anti‐CD163 (Abcam; Ab182422; 1:100) (TAM marker) and mouse anti‐KRT18 (Abcam; Ab668; 1:100) (HPC marker) were used as primary antibodies. Alexa Fluor 488 anti‐mouse IgG (H+L) (Thermo Fisher Scientific; A21202; 1:1000), Alexa Fluor Plus 555 anti‐rabbit IgG (H+L) (Thermo Fisher Scientific; A32732; 1:1000) and Alexa Fluor Plus 647 anti‐rabbit IgG (H+L) (Thermo Fisher Scientific; A32733; 1:1000) were used as secondary antibodies.