Publication protocol
Tissue sections, 5 μm thick, were deposited onto SuperFrost/Plus microscope slides. In order to qualify the material for the study, routine staining of the sections with hematoxylin and eosin (HE) was performed. Anti-human mouse monoclonal antibodies (mAbs) specific for human Ki-67 antigen (clone MIB-1) (Dako Denmark A/S, Glostrup, Denmark, ready to use), anti-p53 (clone DO-7) (Dako), as well as the anti-MUC1 (clone Ma552) and anti-MUC2 (clone Ccp58) (both from NovocastraTM, both in 1:100 dilution) antibodies were used. The sections were incubated with these primary mAbs through the night, at 4 °C, and afterwards with dextran backbone, to which horseradish peroxidase (HRP) was attached, and with secondary biotinylated link anti-rabbit and anti-mouse IgG (Dako REAL™ EnVision™ Detection System peroxidase/DAB+, Rabbit/Mouse, Dako), with microwave-oven pre-treatment for antigen retrieval. Positive reaction manifested, in at least three sequential sections, as a dark brown or black precipitate in the cell nucleus (Ki-67, p53) and cell membrane/cytoplasm (MUC1 and MUC2). The preparations were counterstained using hematoxylin. Every test was accompanied by a negative control, in which specific antibodies were supplemented by a normal serum of a respective species in 0.05 mol/L Tris-HCl, pH approximately 7.6, supplemented with 0.1% bovine serum albumin (BSA) and 15 mmol/L sodium azide (internal negative control). All the steps of immunocytochemistry (IHC) technique were previously described[34]. Histological slides with IHC expression were examined under the optical Olympus BH-2 microscope, coupled to a digital camera. Color microscope images were recorded and archived using a 40 × objective (at least 10 fields in every microscope slide with an IHC positive reaction), with the use of LUCIA Image 5.0 computer software.
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