HSCs or murine tissue sections were fixed with 4% paraformaldehyde followed by permeabilization by 0.2% Triton X-100 and a primary antibody was applied. After incubation with a primary antibody for 2 hours at room temperature or overnight at 4°C, an Alexa Fluor-conjugated second antibody (Thermo Fisher Scientific, Waltham, MA) was used for IF labeling and detection. Cell nuclei were counterstained with DAPI (D1306; Thermo Fisher Scientific). IF signals were captured by an Axio observer (Zeiss, Oberkochen, Germany) with or without ApoTome function.32 (link)To analyze the sorting of TβRII to early endosomes or lysosomes induced by TGFβ1, HSCs expressing TβRII-HA were serum-starved and pretreated with 40 αg/mL of cycloheximide for 1 hour to block the synthesis of proteins including TβRII-HA. TGFβ1 was added in and TGFβ1/receptor binding was permitted by incubating the cells for 30 minutes at 4°C. After washing off free TGFβ1, cells were incubated at 37°C for various times and harvested for double IF for EEA-1/TβRII-HA or LAMP1/TβRII-HA. IF was captured by an Axio observer using a 63x lens with ApoTome function. The rate of TβRII-HA/EEA-1 and TβRII-HA/LAMP1 colocalization was analyzed as we previously described.1 (link)