Identification of Canine Cardiac Mast Cells in Paraffin Embedded Tissue

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Mast cells can be identified in paraffin-embedded canine heart using: (1) toluidine blue staining (see Subheading 3.1.1.); (2) staining with FITC-labeled avidin (Subheading 3.1.2.); and (3) tryptase histochemistry (Subheading 3.1.3.). Fixation is critical for detection of mast cells in canine tissue (see Note 1). Tissue samples are collected from the canine heart and fixed in Carnoy's fixative (60% ethanol, 30% chloroform, 10% acetic acid) for 4 h. Fixed tissues are dehydrated in a graded series of alcohols and embedded in paraffin wax. Sections are cut at 5 dewaxed in xylene, rehydrated, and stained.

3.1.1. Toluidine Blue Staining

Sections stained with toluidine blue are rinsed with 0.5 N HCl (pH 0.5) for 5 min, stained with 0.5 % w/v toluidine blue in 0.5 N HCl for 30 min and

Mast Cell Avidin Fitc

Fig. 2. Identification of mast cells in canine hearts. (A) Staining with FITC-conju-gated avidin identifies mast cells as intensely fluorescent cells packed with granules. (B) In canine infarcts mast cells accumulate in the scar (i) but not in noninfarcted areas (c) (1 h occlusion/7 d reperfusion). (C) Tryptase histochemistry identifies mast cells as red/brown granular cells. (D) Mast cell degranulation in the infarcted myocardium is demonstrated using FITC-avidin staining.

Fig. 2. Identification of mast cells in canine hearts. (A) Staining with FITC-conju-gated avidin identifies mast cells as intensely fluorescent cells packed with granules. (B) In canine infarcts mast cells accumulate in the scar (i) but not in noninfarcted areas (c) (1 h occlusion/7 d reperfusion). (C) Tryptase histochemistry identifies mast cells as red/brown granular cells. (D) Mast cell degranulation in the infarcted myocardium is demonstrated using FITC-avidin staining.

counterstained for 1 min with fast green. Mast cells are easily identified by the dark violet staining of their granules.

3.1.2 FITC-Avidin Staining

Bergstresser et al. (21) described that fluorochrome-conjugated avidin stains rodent and human mast cells. Dewaxed sections are incubated for 60 min at room temperature in FITC-avidin diluted in PBS. The optimal dilution for staining of canine sections fixed in Carnoy's is 1:100. After staining the sections are washed three times for 10 min with PBS and cover slipped with Aquamount without counterstaining. Mast cells are identified as intensely fluorescent granular cells (Fig. 2A,B; see Note 2).

3.1.3 Tryptase Histochemistry

1. To stain for tryptase activity, a reaction mixture is prepared containing 0.54 mM N-CBZ-GLY-GLY-ARG-P-naphthylamide, 0.22 mM Fast Garnet GBC Salt and

3.8% dimethylformamide (by volume) in 50 mM Tris-HCl (pH 6.8) as previously described by Caughey et al. (22).

2. After incubation at 30°C in the reaction mixture for 30 min, the slides are immersed in 1% cupric sulfate for 10 min at room temperature, washed in deionized water for 5 min, and cover slipped with Aquamount without counterstaining.

3. Mast cells are easily identified as red-stained granular cells (Fig. 2C; see Note 3).

3.1.4 Identification of Degranulating Mast Cells in Infarcted Canine Hearts

Although electron microscopy is the gold standard for identifying degranulating mast cells, we have used FITC-avidin staining to demonstrate mast cell degranulation in the canine heart (9). Infarcted hearts show a significant number of degranulating mast cells (Fig. 2D), whereas the majority of mast cells have normal morphology in noninfarcted myocardium.

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