Alexa fluor 488 anti mouse igg antibody

Immunofluorescence staining was performed to assess the constitutive cytosolic double stranded DNA (dsDNA) expression in untreated cells. ID8-Trp53^–/–; Pten^–/– or ID8-Trp53^–/–; Brca1^–/– cells were seeded at a density of 5×10⁵ on coverslips placed in a 6-well plate and left to adhere overnight in complete growth media at 37°C/5% CO₂. Coverslips were washed with phosphate-buffered saline (PBS) and fixed for 10 min with 4% PFA. Following permeabilization of cells (0.2% Triton X-100 in 1× PBS) and blocking (0.1% Triton X-100, 1% bovine serum albumin in 1× PBS) at room temperature (RT), cells were incubated in anti-dsDNA monoclonal antibody (1:100; EMD Millipore, Ontario, Canada) overnight at 4°C. Coverslips were washed with blocking solution three times at RT and incubated in blocking buffer containing Alexa Fluor 488 anti-mouse IgG antibody (1:300; Invitrogen, Massachusetts, USA) for 1 hour at RT in the dark. After counterstaining with DAPI for 10 min, coverslips were rinsed with PBS and mounted with antifading fluorescence medium (Invitrogen, Massachusetts, USA) onto a slide. Slides were imaged using EVOS Cell Imaging System and quantified using ImageJ software.

Cells were fixed in cold methanol for 5 min. After blocking with PBS containing 1% bovine serum albumin and 0.01% Triton X-100 for 1 h at 4 °C, the cells were incubated with anti-LC3B (Sigma-Aldrich Co.) and/or human CD45-FITC antibodies (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) overnight at 4 °C. Bound antibodies were visualized using Alexa Fluor 488 anti-mouse IgG antibody (Invitrogen, Carlsbad, CA, USA). The cells were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained by confocal fluorescence microscopy (Nikon, Tokyo, Japan).