F ab 2 fragment alexa fluor 647 conjugate

Cells were grown on μ‐slides (Ibidi) and fixed in 4% paraformaldehyde (PFA) in D‐PBS (without Ca²⁺ and Mg²⁺) for 10 min at RT followed by PFA inactivation in 300 mM glycine in D‐PBS (5 min, RT) and a wash in D‐PBS. Cells were permeabilized with 1% (v/v) Triton X‐100, 0.2% (w/v) SDS, 10 mg/ml BSA in D‐PBS (1 h, RT) and incubated with primary antibodies overnight at 4°C in 50 mg/ml BSA in TNT (100 mM Tris–Cl (pH 7.5), 150 mM NaCl, and 0.1% (v/v) Tween‐20). The following antibodies and dilutions were used: rabbit anti‐TUJ1 (Cell Signaling, 5568) at 1:600; rabbit anti‐DESMIN (Cell Signaling, 5332) at 1:300; rabbit anti‐GATA6 (Cell Signaling, 5851) at 1:1,600. An anti‐rabbit IgG (H+L) F(ab′)₂ Fragment Alexa Fluor 647 Conjugate (Cell Signaling, 4414) served as secondary antibody and was allowed to incubate for 2 h at RT in 50 mg/ml BSA in TNT. Nuclei were visualized using a constitutive nuclear marker (a stably integrated CAG::H2B‐mCherry‐BGHpA plasmid). Confocal images were acquired on an inverted SP8 confocal microscope (Leica) equipped with a 40× PL Apo 1.1 W objective. TUJ1 immunostaining for quantification by flow cytometry was performed under the same conditions except that the starting material was a single‐cell suspension.

The γH2AX assay was modified on the basis of the previous experimental method [44 (link)]. Approximately 1 × 10⁴ cells were plated into each well of a PhenoPlate 96-well microplate (PerkinElmer, Waltham, MA, USA), and 24 h later cells were continuously exposed to NNK or a combination of NNK and nicotine for 24 h. The cells were then washed with phosphate-buffered saline (PBS) fixed with 4% paraformaldehyde (Beyotime, Shanghai, China), permeabilized with immunostaining permeable fluid (Beyotime, Shanghai, China), and blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA). After blocking, the cells were incubated with 100 μL of 0.1% (v/v) Phospho-Histone H2A.X (Ser139) Antibody (Cell Signaling Technology, Danvers, MA, USA) in 1% BSA overnight at 4 °C. After being washed three times with PBS, the cells were incubated with 100 μL of 0.1% (v/v) Anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor^® 647 Conjugate) (Cell Signaling Technology, Danvers, MA, USA) in 1% BSA for 2 h at room temperature in darkness. Next, 100 μL of Hoechst 33342 (Beyotime, Shanghai, China) was added to each well for 10 min. After the wells were washed with PBS, 100 μL of PBS was added per well and the plates were immediately placed on the PerkinElmer Operetta CLS High Content Screening platform for analysis.