Mouse anti p63 antibody

Cultures were fixed in 4% (w/v) paraformaldehyde for 15 min at room temperature and rinsed in PBS. The samples were dehydrated using a series of increasing ethanol concentrations, cleared with xylene, infiltrated with paraffin wax and embedded in wax blocks. Sections of 2.5 µm thickness were cut using a Thermoshandon Finesse ME + microtome and stained with H&E or PAS using standard histological techniques. For immunohistochemistry, heat-induced epitope retrieval was performed using a Menarini Access Retrieval Unit and staining conducted using a Dako Autostainer. Endogenous peroxidase was blocked with 0.3% (v/v) H₂O₂ in PBS. Basal cells were identified by incubation for 30 min with a 1:30 dilution of mouse anti-p63 antibody (Abcam; #ab735), application of an anti-mouse HRP-labelled polymer and visualization with a REAL EnVision Peroxidase/DAB + Detection System (Dako; #K3468). Samples were subsequently counterstained with Gill’s haematoxylin, dehydrated, cleared and mounted in synthetic resin. Tissue sections were viewed with a Leica DM2000 microscope.

Tracheal epithelial cell cultures were fixed with 0.5 ml 4% (w/v) paraformaldehyde for 15 min. Samples were washed with PBS and dehydrated via a series of increasing ethanol concentrations. The tissues were cleared with xylene and infiltrated with paraffin wax before embedding in wax blocks. Sections of 2.5 μm thickness were cut using a Thermoshandon Finesse ME+ microtome, followed by staining with haematoxylin and eosin (H&E) or periodic acid-Schiff’s (PAS) stain using standard histological procedures. Alternatively, sections were subjected to immunohistochemical labelling by processing with a Menarini Antigen Access Unit. After antigen retrieval, endogenous peroxide was blocked using H₂O₂ in PBS before incubating in mouse anti-p63 antibody (Abcam, #ab735) at a 1 in 30 dilution for 30 min. Primary antibody was labelled using an anti-mouse-HRP-labelled polymer and detected using a REAL EnVision Peroxidase/DAB+ Detection System (Dako, #K3468) according to the manufacturer’s instructions. Sections were counter-stained using Gill’s haematoxylin and dehydrated, cleared and mounted in synthetic resin. Visualisation was carried out using a Leica DM2000 microscope.