After deparaffinization and rehydration, tissue sections (2.5 μm) were also submitted to immunohistochemical assay. Antigenic recuperation was performed by heat in citrate pH 6.0 solution. After cooling, the slides were submitted to peroxidase blocking with H2O2 3% solution diluted in PBS (phosphate buffered saline) for 30 minutes.
After protein blocking (PBS) for 1 hour, the specimens were incubated overnight with Tumor Necrosis Factor alpha (TNF-α) (Abcam®, Cambridge, UK) in 1:100 dilution. Then, the primary antibody Simple Stain Rat MAX PO (Multi) Universal Immuno-peroxidase Polymer (anti-mouse and -rabbit) (Histofine®, Nicherei Biosciences Inc., Tokyo, Japan) was used for 60 minutes. The revelation system 5,5-diaminobenzidine tetrahydrochloride (DAB) (Dako®, Carpinteria, CA, USA) was used for 5 minutes and the counter coloration was Harris hematoxylin, which happened for 30 seconds.
The percentage of cells in connective tissue with cytoplasmatic or nuclear expression was grouped into scores: (0) no positive cells; (1 - mild) 1-33% of positive cells; (2 - moderate) 34-66% of positive cells; (3 - intense) 67-100% positive cells. The same score, obtained by two or more observers, was considered as the final score11 .
After protein blocking (PBS) for 1 hour, the specimens were incubated overnight with Tumor Necrosis Factor alpha (TNF-α) (Abcam®, Cambridge, UK) in 1:100 dilution. Then, the primary antibody Simple Stain Rat MAX PO (Multi) Universal Immuno-peroxidase Polymer (anti-mouse and -rabbit) (Histofine®, Nicherei Biosciences Inc., Tokyo, Japan) was used for 60 minutes. The revelation system 5,5-diaminobenzidine tetrahydrochloride (DAB) (Dako®, Carpinteria, CA, USA) was used for 5 minutes and the counter coloration was Harris hematoxylin, which happened for 30 seconds.
The percentage of cells in connective tissue with cytoplasmatic or nuclear expression was grouped into scores: (0) no positive cells; (1 - mild) 1-33% of positive cells; (2 - moderate) 34-66% of positive cells; (3 - intense) 67-100% positive cells. The same score, obtained by two or more observers, was considered as the final score