Lsab2 dako kit

Tissue specimens from central bronchi were selected, and fixed with 10% neutral buffered formalin and embedded in paraffin wax. Sequential sections (3 µm thick) were placed on poly-L-lysine coated slides, deparaffinised in xylene, rehydrated in a descending ethanol series and stained with haematoxylin and eosin (HE). Immunoreactivity for EZH2, H3K27me3and DAB2IP were observed using: a mouse monoclonal Ab anti-EZH2 (clone 144CT2.1.1.5) (Thermo Fisher Scientific, Rockford, USA), Polyclonal Antibody Anti-Trimethyl-Histone H3 (Lys27) (Cat#07-449 EMD Millipore Corporation, Temecula, CA, USA) and a rabbit Polyclonal Abanti-DAB2IP (ab87811, Abcam, Cambridge, UK) in Higher airways (internal perimeter >6 mm) by Immunohistochemistry²⁰ . LSAB2 Dako kit (Code N° K0674) (Dako, Glostrup, Denmark) and Fuchsin Substrate-Chromogen System Dako²¹ were used for the staining. Rabbit and mouse immunoglobulins (Dako) were used for negative controls. Two independent investigators, using image analysis (Leica microscope, Wentzler, Germany) 40X magnification, evaluated sample immunoreactivity blindly. The length of the basement membrane was evaluated using a Leica Application Suite V3.3 (LAS) software (Leica) for Image Analysis. Results were expressed as the number of positive epithelial cells/mm basement membrane as previously described²² .

Sections (5 μm thick) of paraffined-embedded brains were hydrated in a sequence of graded ethanol (from 96% to 50%) for 5 min each, washed in water and then PBS. The slides were incubated with 3% BSA for 1 h and subsequently, with anti-CD4+ (1:40) (Dako) at 4 °C overnight. After washing, the LSAB2 Dako Kit (Dako) and Fuchsin Substrate-Chromogen System (Dako) were used for brown staining. The slides were mounted with cover slips, and images were visualized using a Leica DM5000 upright microscope (Leica Microsystems) at a magnification of 20×.