Alexa fluor 633 conjugated goat anti rabbit antibody

Mice were anesthetized with ketamine/xylazine and perfused transcardially with Ringer’s solution and 4% paraformaldehyde in PBS. Immunofluorescence was performed in 40-μm-thick free-floating sections, blocking the tissue in 4% normal goat serum, 0.05% Triton TX-100 (Sigma-Aldrich), and 4% bovine serum albumin (Sigma-Aldrich) in PBS. Sections were incubated overnight at room temperature with the primary anti-Iba1 rabbit antiserum (1:1000; Wako, Osaka, Japan), and antibody binding was detected using an Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1000; Invitrogen). Sections were finally stained with DAPI (1:50,000; Sigma-Aldrich), mounted on glass slides in a 0.2% solution of gelatin in 0.05 M Tris-HCl buffer, and dried and then dehydrated in xylene (Panreac, Barcelona, Spain) for 12 min before coverslipping in DPX (VWR, Leuven, Belgium). Tissue sections were visualized in a confocal laser scanning microscope LSM800 with Airyscan (Zeiss, Oberkochen, Germany). Images were acquired using a × 63 oil objective with constant microscope parameters and laser intensity. A projection stack of 10 images per slice is shown.

Confocal microscopy was performed to evaluate the effect of adavosertib on mitosis. DTC cells were plated for overnight at 5 × 10⁴ cells (BHP7-13, FTC-238) and 1 × 10⁵ cells (K1, FTC-133) in 4-well culture slides in 1 mL of media, and were incubated for 48 h (BHP7-13, K1, FTC-133) and 24 h (FTC-238) in the presence of adavosertib (500 nmol/L) or placebo. The cells were then washed with PBS, fixed with 4% paraformaldehyde (Merck) for 15 min at room temperature, washed with PBS, permeabilized with 0.1% Triton X-100 for 10 min, and washed with PBS. The samples were then probed with primary rabbit p-Histone H3 (Ser10) antibody (1:200) and mouse α-tubulin antibody (1:1000) at 4 °C overnight in a humidified chamber. The slides, stained in the dark with secondary Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1000; Invitrogen) and Alexa Fluor 488-conjugated goat anti-mouse antibody (1:1000; Life Technologies, Carlsbad, CA, United States) for 25 min at 37 °C, were washed with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI; 0.2 μg/mL) (Invitrogen) for 10 min at room temperature, covered with mounting medium. Chromosomes were examined to identify mitotic cells. We also identified thyroid cancer cells with p-Histone H3 staining. Samples were imaged with a Leica TCS SP8 X confocal microscope (Leica Microsystems, Wetzlar, Germany).

Mouse antibody against Flag-tag (M2) and M2-conjugated agarose were purchased from Sigma-Aldrich (St. Louis, MO); rabbit antibodies against TOM20 and the p65 subunit of NF-κB were from Santa Cruz Biotechnology (Dallas, TX); Alexa Fluor 488 conjugated goat anti-mouse and anti-rabbit antibodies, Alexa Fluor 568 conjugated goat anti-mouse antibody, and Alexa Fluor 633 conjugated goat anti-rabbit antibody were from Invitrogen (Carlsbad, CA). Sendai virus (SeV, Cantell strain, Charles River Laboratories) was used at 100 hemagglutination (HA) units/ml culture media. HEK293T, HEK293T-IFNβ-luciferase, Mavs^−/− MEF cells and derivatives were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) cosmic calf serum (Hyclone, Thermo Fisher Scientific, Waltham, MA) with penicillin (100 U/ml) and streptomycin (100 μg/ml). Other chemicals and reagents were from Sigma-Aldrich unless otherwise specified.