Histochemical Staining of Tissue Samples

Formalin-fixed tissue samples were dehydrated and embedded in paraffin. Tibiotarsal joints were decalcified before they were embedded in paraffin. The tissues in the paraffin sections (1 to 2 μm) were dewaxed, hydrated, and histochemically stained with hematoxylin and eosin (Merck) to obtain an overview, toluidine blue to observe mast cells, and a modified Sirius red staining protocol to observe eosinophil granulocytes (35 (link)). Sections were stained with toluidine blue (Sigma) for 10 min, washed with distilled water, and dehydrated by dipping them in 1% acetic acid and 100% ethanol. Sections were cleared in xylol, and coverslips were mounted with Corbit balsam (Hecht). For staining of eosinophils, sections were stained for 1 min with hematoxylin (Merck), washed with tap water, and dehydrated with 100% ethanol before staining with direct red 80 dye (Sigma) for 2 h. Sections were washed with tap water, dehydrated with 100% ethanol, and cleared in xylol before coverslips were mounted with Corbit balsam. Images were acquired using a fluorescence microscope (AxioImager Z1) equipped with a charge-coupled-device camera (AxioCam MRm) and processed with Axiovision software (Carl Zeiss AG, Germany).