Goat serum

Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with PBS containing 0.1% (v/v) Triton X-100 for 10 min. The cells were blocked with 5% goat serum and 1% BSA (Wako) in TBS-T for 60 min, incubated with appropriate primary antibodies overnight at 4 °C, and then incubated with an appropriate secondary antibody and DAPI. Stained cells were visualized by high-resolution confocal microscopy (SP8; Leica).

Cells were cultured on cover glass, fixed with 4%-Paraformaldehyde Phosphate Buffer Solution (nacalai tesque), and permeabilized with 0.5% (vol/vol) Triton X-100 (nacalai tesque) in PBS, followed by incubation in blocking solution (2% (vol/vol) goat serum (FUJIFILM Wako Pure Chemical) and 0.1% (wt/vol) gelatin in PBS). The antibodies used in Immunofluorescence were listed in the key resources table. DNA was stained with Hoechst 33342 (Invitrogen). Fluorescence was detected with TCS SPE (Leica). Acquired images were analyzed with Fiji (Schindelin et al., 2012 (link)).

Human brain VSMC grown on coverslips were fixed in 4% PFA in PBS for 20 min at room temperature. After rinsing in PBS, cells were permeabilized with 0.1% Triton X-100 for 5 min, followed by blocking in 5% goat serum (Wako, 143-06561) for 30 min at room temperature. Autophagosomes, α-SMA, and vimentin were visualized by incubating cells for 60 min at room temperature with rabbit monoclonal anti-LC3A/B (1:500, Cell Signaling, #12741), rabbit monoclonal anti-α-SMA (1:200, Abcam, ab32575), and rabbit monoclonal anti-vimentin (D21H3) XP (1:200, Cell Signaling, #5741), respectively. In all immunocytochemisty experiments, cells were double-stained with mouse monoclonal anti-LAMP-2/CD107b antibody (1:200, Novus Biologicals, NBP2–22217) for estimation of LAMP-2 siRNA transfection efficiency. Nuclei were counterstained using DAPI and coverslips were mounted with Prolong Gold antifade reagent (Invitrogen, P36930).

For histological analysis, 0.5 cm distal colon was fixed in Zinc Formalin (Polyscience Inc.) for 3 hours and then embedded in paraffin blocks. We then prepared 5 μm paraffin cross-sections, which were used for hematoxylin and eosin (H&E) staining (Mayer’s Hematoxylin solution, 1% Eosin Solution, Wako) following standard procedures. The histological images were captured using the BX51-P Polarizing microscope (Olympus) and processed with the Olympus D.P. Controller 2002 software.
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).

ARPE-19 cells incubated for 24 h after supernatant transfer in 35-mm dishes were fixed with 100% methanol (Wako, Osaka, Japan) for 5 min at room temperature, and incubated with PBS containing 10% goat serum (Wako, Osaka, Japan), 0.3 M glycine (Wako, Osaka, Japan), 1% bovine serum albumin (Wako, Osaka, Japan) and 0.1% tween (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h at room temperature. The cells were then incubated with 1 μg/mL of rabbit anti-Claudin-1 or anti-ZO-1 antibody overnight at 4 °C, and with 1 μg/mL of goat anti-rabbit IgG H&L for 1 h at room temperature with 1 μg/mL of Hoechst 33342. Fluorescence images of the cells were obtained with a fluorescence microscope (KEYENCE, Osaka, Japan).

pGCTB-SCs were seeded on poly-L-lysine (Fujifilm Wako)–coated cover glass at a density of 10 × 10⁴ cells and cultured for 48 h. After the cells were treated with each reagent for the indicated periods, they were fixed with 4% paraformaldehyde (FUJIFILM Wako) for 10 min at RT, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and blocked with 10% goat serum (FUJIFILM Wako) for 30 min. Subsequently, the cells were incubated at 4 °C overnight with a mixture of primary antibodies diluted in 1:200 in Can Get Signal Immunostain Solution A (TOYOBO, Osaka, Japan). Samples were then washed three times with PBS and incubated with Alexa Fluor® 488 and 594 diluted in 1:200 for 1 h at RT. SlowFade Diamond antifade mountant with DAPI (Invitrogen) was used as a mounting solution. Immunostaining was visualized using fluorescence microscopy (BZ-X800; Keyence).