Mouse monoclonal anti mast cell tryptase antibody

The rats were sacrificed after the experiment. For histological analysis, a 1-cm length specimen of the distal colon was obtained from each rat and fixed using 10% buffered formalin. The paraffin embedded specimens were sectioned perpendicularly to the lumen (section thickness, 4 μm), and mounted on a slide glass. The immunohistochemistry (IHC) stain was done by using an automatic immunostainer (BenchMark XT; Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. The negative IHC control omitted the primary antibody. The slides were incubated with primary antibodies and a mouse monoclonal anti-mast cell tryptase antibody (Abcam Inc, Cambridge, MA, USA). Sections were then counterstained with hematoxylin for 4 minutes to stain the nucleus and then dehydrated, cleared, and mounted in synthetic mountant. Photographs of tryptase positive cells were obtained from 6 to 8 non-overlapping fields on 2 immunostained slides per rat under a light microscope (Carl Zeiss, Jena, Germany) linked to a computer-assisted image analysis system. The number of cells stained with the primary antibody was counted in all the photographs by the 3 researchers who were blinded to the animal groupings, and the cell numbers were expressed as visible mast cells in each high-power field (number of cells/hpf).