Alexa fluor 594 goat anti rabbit antibody

293T (ATCC ref. CRL-3216) and Ad-293 (Agilent Technologies ref. 240,085) cells were grown in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. For immunofluorescence experiments, 293T cells were grown on collagen-coated coverslips and transfected with 1 μg/ml plasmid DNA (expressing N- or C-terminally Flag-tagged, or untagged Mustn1) using 5 μg/ml polyethylenimine (PEI MAX, Polysciences ref. 24,765). 24 h after transfection, cells were fixed with 4% paraformaldehyde in 0.1M phosphate buffer (PBS) for 15 min, washed three times with PBS, permeabilized for 15 min (0.25% Triton X-100 in PBS), and incubated with blocking solution (10% goat normal serum in PBS) for 15 min, all at room temperature. Cells were then incubated with rabbit anti-Mustn1 antibody (Merck Millipore, ABD115, 1:500) overnight at 4 °C, washed three times with PBS, followed by incubation with AlexaFluor 594 goat anti-rabbit antibody (Invitrogen, A11037, 1:500) for 1 h at room temperature. Both primary and fluorophore-conjugated secondary antibodies were diluted in blocking solution. Cells were washed three times with PBS, counter-stained with DAPI and mounted on a microscopy slide using Vectashield for imaging. Images were captured using a Zeiss Axio Vert.A1 widefield fluorescence microscope with a LD Plan-Neofluar 40x/0.6 Korr M27 objective.

Mice were anesthetized with sodium pentobarbital and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PBS) after the probe trial. Brains were stored in 4% paraformaldehyde at 4 °C overnight then dehydrated in 30% phosphate-buffered sucrose solution for cryoprotection. Samples were preserved at − 80 °C and coronal sections of 20 µm were obtained by a cryostat (Leica Microsystems, Wetzlar, Germany). Sections were incubated overnight at 4 °C with the rabbit anti-GFAP (1:2000; Dako, Glostrup, Denmark) primary antibody, and sequentially incubated for 2 h with Alexa Fluor 594 goat anti-rabbit antibody at room temperature (1:500; Invitrogen, Eugene, OR, USA). Staining of β-Amyloid plaques was performed using Thioflavin S (ThS 0.002%, Sigma-Aldrich) to compare β-amyloid plaque density among different treatment groups. Sections were counterstained with 0.1 μg/ml Hoechst 33,258 (Sigma-Aldrich, St Louis, MO, USA) and rinsed afterwards with PBS 0.1 M [30 ]. ThS-stained β-amyloid plaques were visualized using a fluorescence microscope with a fluorescence filter (BX41 Laboratory Microscope, Melville, NY-Olympus America Inc). For each image, the proportion of total image area covered by fluorescently stained β-amyloid plaques was quantified. For each mouse, four fields per section with the highest density of plaques were chosen as representative and were averaged [31 (link)].

Rabbit polyclonal anti‐gamma H2AX antibody (ab2893), rabbit monoclonal anti‐Cyclin B1 antibody (ab181593), mouse monoclonal anti‐CDK1 antibody (ab18), and sheep polyclonal anti‐BUBR1 antibody (ab28193) were from Abcam (Cambridge, UK). Rabbit polyclonal anti‐MAD2 antibody (10337‐1‐AP) and CoraLite594‐conjugated donkey anti‐Rabbit IgG (H + L) (SA00013‐8) were from Proteintech (Wuhan, China). Alexa Fluor 594 goat anti‐rabbit antibody, Alexa Fluor 488, and 594 goat anti‐sheep antibody were from Invitrogen (Carlsbad, CA). Rabbit polyclonal anti‐centromere (15‐234‐0001) was from Antibodies Incorporated (Shenzhen, China). Horseradish peroxidase‐conjugated goat anti‐rabbit/mouse IgG antibodies were from Beyotime (Nantong, China). Mouse monoclonal anti‐α‐tubulin‐FITC antibody (F2168), Hoechst 33342 (B2261) and all other unstated chemicals were from Sigma (St. Louis, MO).

Immediately after the Raman measurements, the HepaRG cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and stored at 4 °C until further analysis. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 4% bovine serum albumin (A2153; Sigma-Aldrich) for 1 h at RT. A primary antibody working solution containing mouse anti-CYP3A4 (1:1000; SAB5300118; Sigma-Aldrich) and rabbit anti-human cyt b₅ (1:500; ab69801; Abcam) was prepared in 1% bovine serum albumin solution, applied to the cell samples, and incubated overnight at 4 °C. The following day, the samples were washed three times with PBS and then immersed in a secondary antibody solution containing Alexa Fluor 488 goat anti-mouse antibody (10 µg/mL; A11001; Invitrogen) and Alexa Fluor 594 goat anti-rabbit antibody (10 µg/mL; A11012; Invitrogen) for 1 h at RT. After washing with PBS three times, the cells were counterstained with 1 µM DAPI solution (D1306; Invitrogen) at RT. The cells were then washed twice with PBS, and the dishes were stored and protected from light at 4 °C until image acquisition. Fluorescent images were captured using a confocal laser scanning microscope (A1; Nikon).

The CHK2 inhibitor BML-277 was purchased from Merck and Millipore (USA). Rabbit polyclonal anti-CHK2 antibody, rabbit monoclonal anti-γ-H2A.X antibody and anti-MAP1LC3A antibody were purchased from Abcam (Cambridge, UK). Anti-α-tubulin-FITC antibody and Hoechst 33342 were purchased from Sigma (St. Louis, MO, USA). Alexa Fluor 488 goat anti-rabbit antibody and Alexa Fluor 594 goat anti-rabbit antibody were purchased from Invitrogen (Carlsbad, CA, USA).