Real envision peroxidase dab detection system

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.

ALI cultures were fixed in 4% (w/v) paraformaldehyde and stored in PBS. Cultures were processed by dehydrating through a series of increasing ethanol concentrations, cleared with xylene, infiltrated with paraffin wax and embedded in wax blocks. Sections (2.5μm in thickness) were cut using a Thermoshandon Finesse ME+ microtome and were stained with hematoxylin and eosin (H&E) or Periodic Acid Schiff (PAS) stain according to standard histological techniques. For immunohistochemistry (IHC), sections were subjected to antigen retrieval using a Menarini Access Retrieval Unit. Endogenous peroxidase was blocked using H₂O₂ in PBS. Sections were subsequently incubated with mouse anti-p63 antibody (Abcam #ab735) at a 1:30 dilution for 30 min followed by the application of an anti-mouse HRP-labelled polymer, before visualization with a REAL EnVision Peroxidase/DAB+ Detection System (Dako #K3468) according to manufacturer’s instructions. Samples were counterstained with Gill’s hematoxylin before dehydration, clearing and mounting in synthetic resin. Slides were visualized using a Leica DM2000 microscope.