Primary antibody diluting buffer

For the immunofluorescence, eye sections were deparaffinized and rehydrated before being incubated overnight in Epitope Retrieval Solution (Leica Biosystems Inc., Buffalo Grove, IL, USA) at 60 °C. Then, the sections were blocked at room temperature for 40 min with a solution containing PBS, 2% bovine serum albumin (BSA) (ABIN934476, Antibodies-online), and 0.1% Triton-X100 (X100, Sigma-Aldrich, St. Louis, MO, USA). Primary rabbit anti-VEGFR2 (bs-10412R, 1:100 dilution; Bioss, Woburn, MA, USA) and rabbit anti-TACE (bs-4236R; 1:100 dilution; Bioss, USA) antibodies were diluted in the primary antibody diluting buffer (Bio-Optica, Milano, Italy) and incubated for 2 h at room temperature. Slides were incubated for 30 min at room temperature in the dark with Cy5-labeled goat anti-rabbit IgG secondary antibody (A10523; Thermo Fisher Scientific Inc., Rockford, IL, USA) and diluted 1:500 in PBS before being counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. CC/Mount aqueous mounting medium (Sigma-Aldrich, St. Louis, MO, USA) was used to mount the slides. Images were acquired using a Leica TCS SP8 laser scanning confocal microscope. The percentage of positive-stained area/total area was quantified using ImageJ software.

For immunofluorescence, the deparaffinized, rehydrated liver sections were incubated overnight in epitope retrieval solution (Leica Biosystems Inc., Buffalo Grove, IL, USA) at 60 °C, followed by blocking with PBS containing 2% bovine serum albumin (BSA) (ABIN934476, Antibodies-online) and 0.1% Triton X-100 (X-100, Sigma-Aldrich, St Louis, MI, USA) at room temperature for 40 min. The tissue sections were incubated with rabbit anti-Smad 2/3 (sc-8332, 1:300 dilution; Santa-Cruz, CA, USA) and rabbit anti-α-SMA (ab32575; 1:500 dilution; Abcam, Cambridge, UK) primary antibodies for 2 h at room temperature. The primary antibodies were diluted in primary antibody diluting buffer (Bio-Optica, Milano, Italy). The slides were washed in PBS three times and incubated with the Alexa Fluor 594 labeled goat anti-rabbit IgG secondary antibody (A 11037; Thermo Fisher Scientific Inc., Rockford, USA), diluted to 1:400 in PBS, for 30 min at room temperature in the dark. After three more washing steps with PBS, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, washed by PBS, and mounted with CC/Mount aqueous mounting medium (Sigma-Aldrich, St Louis, MI, USA). Images were captured using a Leica TCS SP8 laser scanning confocal microscope.

GFAP levels were assessed by using a rabbit polyclonal anti-GFAP antibody (ab7260; Abcam PLC., Cambridge, UK) and AlexaFluor 594 labeled goat anti-rabbit IgG secondary antibody (A 11037; Thermo Fisher Scientific Inc., Rockford, IL, USA). Goat polyclonal Iba-1 (ab-5076; Abcam PLC., Cambridge, UK) and donkey anti-goat AlexaFluor 594 (a-11058; Invitrogen, Waltham, MA, USA) were used as primary antibody and secondary antibody, respectively. Bond Dewax Solution (Leica Biosystems Inc., Buffalo Grove, IL, United States) was used to deparaffinate the eye sections. They were rehydrated in alcoholic solutions. Epitope Retrieval Solution (Leica Biosystems Inc., Buffalo Grove, IL, United States) was used for antigen retrieval at 95 °C for 10 min, followed by blocking with 2% BSA in PBS. The primary antibody was applied in a dilution of 1:1000 in primary antibody diluting buffer (Bio-Optica, Milano, Italy) for 2 h at 4 °C. The slides were washed in PBS and incubated with the secondary antibody, diluted to 1:500 in PBS, for 2 h at room temperature in the dark. After a further 3 washing steps with PBS, nucleus counterstaining was performed with 1 μg/mL DAPI (Sigma-Aldrich, St Louis, MO, USA). CC/Mount aqueous mounting medium (Sigma-Aldrich, St Louis, MO, USA) was used to mount the stained slides. They were examined with a Leica SP5 confocal laser scanning microscope.