Epr1473 to cdkn2a p16ink4a tag

Endometriosis lesion, non-endometriosis, and eutopic endometrial tissue samples of 4 µm thickness were prepared and mounted on slides following an immunofluorescence protocol for co-localization of p16^Ink4a and lamin b1 with e-cadherin (epithelial cell marker). Briefly, samples were thawed, fixed in 4% paraformaldehyde, washed in PBS with 0.5% Tween, and immersed in Triton X-100 for better antibody perfusion. Subsequently, samples were incubated overnight with a monoclonal antibody against p16^Ink4a (1:100, rabbit monoclonal (EPR1473) to CDKN2A/p16^INK4a tag; Abcam, Cambridge, UK) or a polyclonal antibody against lamin b1 (1:100, H90, catalog no. Sc-20682; Santa Cruz Biotechnology, Dallas, TX, USA) and mixed with pre-diluted e-cadherin (Ref. GA059—Monoclonal mouse anti-human e-cadherin clone NCH-38 ready-to-use (Dako Omnis), Santa Clara, CA, USA). The slides were then washed in PBS with 0.5% Tween, and both secondary antibodies, Alexa Fluor^®® 488 (1:400, Goat Anti-Rabbit IgG H&L (ab150077) Abcam, Cambridge, UK) and Alexa Fluor^®® 594 (1:400, Goat Anti-Mouse IgG H&L pre-adsorbed (ab150116) Abcam, Cambridge, UK), were added. A qualitative analysis was performed using a Carl Zeiss LSM 710 confocal microscope (Oberkochen, Germany) fitted with Zen (2010) software (Zeiss, Oberkochen, Germany).

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.