Quantitative Immunohistochemical Analysis of Protein Acetylation

Serial sections from TissueTek-embedded biopsy samples were cut with a cryostat (5 μm), fixed with acetone and endogenous peroxidase activity blocked with 0.3% hydrogen peroxide in 0.1% sodium azide/phosphate-buffered saline.^{27 (link)} Sections were stained overnight at 4°C with rabbit antibodies against acetylated lysine (acLys) or acetylated histone 3(Lys18) (acH3) (both from Cell Signaling Technology). Equivalent concentrations of control rabbit antibodies (anti-fluorescein isothiocyanate, Thermo Scientific) were applied for control sections. Sections were then washed and incubated with horseradish peroxidase (HRP)-conjugated swine anti-rabbit antibodies (Dako), followed by incubation with biotinylated tyramide and streptavidin-HRP, and development with amino-ethylcarbazole (AEC, Vector Laboratories). Sections were subsequently counterstained with Gill’s haematoxylin (Klinipath) and mounted in Kaiser’s glycerol gelatine (Merck). For quantitative analysis of protein acetylation, stained sections were randomly coded by an independent observer, blinded to antibodies used and clinical diagnosis. Stained slides were analysed by computer-assisted image analysis using the Qwin analysis system (Leica) as previously described.^{28 (link)} Values of integrated optical densities (IOD)/mm² were obtained and corrected for total number of nucleated cells per square millimetre.