Alexa 488 conjugated secondary antibody

Tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed using 1% Triton X in PBS for 10 min. Immunofluorescence staining was performed by incubation with a phospho-specific p65 antibody (#3033, cell signaling, 1:50) overnight. Slides were subsequently washed and incubated with an Alexa 488-conjugated secondary antibody (#4412, Cell Signaling, Cambridge, UK, 1:100), counterstained with nuclear DAPI (1 µg/ml, #10236276001, Sigma-Aldrich, St. Louis, USA) and mounted with fluorescent mounting medium (#S3023, Dako, Santa Clara, USA).

The cultured cells mentioned above were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), followed by cellular permeabilization with 0.01% Triton-X 100 and 30 min of incubation between 22 and 24 °C in a blocking solution comprising PBS with 1% bovine serum albumin. Subsequently, the cells were incubated for 14–16 h with primary antibodies against vimentin (1:200, catalog no. Sc-373717; Santa Cruz Biotechnology, Dallas, TX, USA), E-cadherin (1:200, Dako Agilient, Santa Clara, CA, USA), p16^ink4a (1:100, rabbit monoclonal (EPR1473) to CDKN2A/p16INK4a tag; Abcam, Cambridge, UK), and lamin B1 (1:50, H90, catalog no. Sc-20682; Santa Cruz Biotechnology), in separate wells. The Alexa 488-conjugated secondary antibody (Cell Signaling, Beverly, MA, USA) and Alexa Fluor 694-conjugated antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA) were used as secondary antibodies (1:400 dilution). Nuclear staining was performed using Vectashield medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Cells were also incubated with Cell Trace Violet Kit—cell proliferation (C34557—Thermo Fisher Scientific, Waltham, MA USA) for morphology characterization. Cell images were acquired with an LSM710 confocal microscope (Zeiss, Jena, Germany). HeLa cells were used as positive controls for p16^ink4a and lamin B1 staining.