Hydromount solution

Cells were seeded on 12-mm glass coverslips and fixed for 15 min with 4% paraformaldehyde. Non-specific binding sites were blocked by incubation with TBS containing donkey serum (10%) for 1 h and coverslips were then incubated overnight at 4 °C with the following antibodies diluted in TBS 1X: goat polyclonal anti-CR (1:500; cat# CG1, Swant, Marly, Switzerland) and rabbit polyclonal anti-septin 7 (1:500; Bethyl Laboratories, USA). After washing, coverslips were incubated with secondary antibodies for 3 h at room temperature with the following secondary antibodies: Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:100, Jackson Immunoresearch Laboratories, West Grove, PA, USA) and Cy5-conjugated donkey anti-goat IgG (1:100; Jackson). Nuclear DNA was stained using DAPI (5 μg/ml; Molecular Probes, Eugene, OR) and coverslips were mounted with Hydromount solution (National Diagnostics, Atlanta, GA). Images were acquired using a Leica fluorescent microscope DM6000B (Wetzlar, Germany) equipped with a Hamamatsu camera C4742–95 (Bridgewater, NJ). For the cells treated with Bt, cells were seeded onto 12-mm glass coverslips pre-coated with Matrigel (Corning, NY, USA) and treated with 1 mM Bt for 48 h.

Cells were seeded on glass coverslips and incubated with the appropriate cell culture medium until a confluence of 70–90% was reached. Then, the cells were fixed with 4% PFA, permeabilized with 1% Triton X-100, and blocked with TBS solution containing donkey serum (10%). Cells were incubated overnight at 4°C with the primary antibodies at the indicated dilutions: mouse monoclonal anti-pan-cytokeratin, clone Lu-5 (BMA Biomedicals, Augst, Switzerland, T-1302; 1:500), or rabbit polyclonal anti-desmin (Sigma D8281; 1:500). After washing, the cell-containing coverslips were incubated with secondary antibodies for 1 h with either DyLight488-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA; 1:1,000) or Cy3-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories; 1:1,000). The cells were counterstained with DAPI (Molecular Probes, Eugene, OR; 5 μg/ml) and mounted with Hydromount solution (National Diagnostics, Atlanta, GA). Images were acquired with a LEICA fluorescent microscope DM6000B (Wetzlar, Germany) equipped with a Hamamatsu camera C4742-95 (Bridgewater, NJ).

SPC212 cells were seeded on glass coverslips in 24-well plates. After fixation for 15 min with 4% paraformaldehyde (37°C), non-specific binding sites were blocked by incubation with TBS containing donkey serum (5%) for 1 h. The rabbit polyclonal anti-septin 7 antibody (Bethyl Laboratories, Montgomery, TX, USA) was diluted 1:2,500 in TBS and coverslips were then incubated overnight at 4°C. After washing, the secondary antibody (Alexa Fluor 488-conjugated donkey anti-rabbit IgG 1:400, Jackson Immunoresearch Laboratories, West Grove, PA, USA) was added for 3 h at room temperature. Nuclei were stained with Hoechst 33342 (1μg/ml, 10 min; Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Next, the coverslips were mounted with Hydromount solution (National Diagnostics, Atlanta, GA). A Leica TCS SP5 confocal microscope with a 63x glycerol-immersion objective (1.3 NA, Plan APO) was utilized for the image acquirement.

Twenty-micrometer coronal brain sections from −1.70 mm from the bregma were selected, and standard immunostaining techniques were employed. Briefly, 20-μm sections were washed three times with 1x PBS, blocked for 1 h in goat serum containing 0.4% Triton X-100, and incubated overnight at 4 °C with primary antibody (rabbit anti-P2Y12 (1:1000, AnaSpec Inc., Fremont, CA; catalog no: AS-55043A), rabbit anti-Iba-1 (1:200; Wako Chemicals, Richmond, VA; catalog no: 019-197)). Sections were washed three times with 1x PBS and incubated with appropriate Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 2 h at RT. Sections were washed three times with 1x PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml, Sigma), and mounted with glass cover slips using Hydromount solution (National Diagnostics, Atlanta, GA). Images were acquired using a fluorescent Nikon Ti-E inverted microscope, at ×10 (Plan Apo 10× NA 0.45) or ×20 (Plan APO 20× NA 0.75) magnification. Exposure times were kept constant for all sections in each experiment. All images were quantified using Nikon ND-Elements Software (AR 4.20.01). 6000–10,000 positive areas were quantified per mouse per experiment, and expression levels were expressed as binary area per region of interest (ROI) × 10⁶.