Alexa fluor plus 488 conjugated goat anti rabbit immunoglobulin g

Immunofluorescence staining was performed by seeding cells at a density of 5 × 10⁴ cells/well on a 15 mm poly-L-lysine-coated cover glass in a 6-well plate, followed by a 24 h incubation at 37 °C. The cells underwent fixation with 4% paraformaldehyde, permeabilization with 0.1% Triton X-100 for 15 min at room temperature, and triple washing with phosphate-buffered saline (PBS). Subsequently, cells were blocked with 1% bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA) for 60 min at room temperature. Following three 10 min washes with PBS, the samples were incubated overnight at 4 °C with an anti-BRD4 monoclonal antibody. After three 3 min PBS washes, cells were exposed to Alexa Fluor Plus 488 conjugated Goat anti-rabbit immunoglobulin G (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at room temperature. Mitochondria were stained with MitoTracker Orange (1:5000, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at room temperature. Visualization and photography of fluorescence-labeled HOXB9 and mitochondria were conducted using a fluorescence microscope.

The cells were seeded at 3 × 10⁴ cells/well onto 15 mm poly-L-lysine coated cover glass in a 24-well plate and incubated at 37 °C for 24 h. The cells were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 for 15 min at RT, and subsequently, washed with phosphate-buffered saline (PBS) 3 times for 3 min each. Following this, cells were blocked with 1% bovine serum albumin (Cat# A7030, Sigma Aldrich, St. Louis, MO, USA) for 60 min at room temperature. After the samples were washed with PBS 3 times for 10 min, the samples were incubated with anti-HOXB9 monoclonal antibody (1:500, Cat# 27967, Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4 °C. Subsequent to 3 washes for 3 min each with PBS, the cells were incubated with Alexa Fluor Plus 488-conjugated Goat anti-rabbit immunoglobulin G (1:1000, Cat# A32731, Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at room temperature. The mitochondria were stained with MitoTracker Orange (1:5000, Cat# M7511, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at room temperature. Fluorescence-labeled HOXB9 or mitochondria were observed and photographed under a fluorescent microscope.