Exorbital lacrimal and submandibular salivary glands were harvested and fixed in buffered formalin, processed, embedded in paraffin, and sectioned. Five micrometer sections of paired glands were stained with hematoxylin and eosin (H&E) and analyzed by standard light microscopy. Inflammation was quantified using standard focus scoring57 (link). Focus scores (number of inflammatory foci per 4 mm2) were calculated by a blinded observer by counting the total number of foci (composed of ≥ 50 mononuclear cells) by standard light microscopy using a 10× objective, scanning slides to obtain digital images using PathScan Enabler IV (Meyer Instruments), and measuring surface area of sections using ImageJ software58 (link). Samples with diffuse inflammation resulting in coalescence of individual foci were assigned focus score values greater than the highest calculable value for that set of comparisons. Representative images were captured on a Leitz DM-RB research microscope with a Leica DCF700T digital camera using Leica Application Suite X software (Leica Microsystems, Wetzlar, Germany).
Protocol detail
Quantification of Glandular Inflammation
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Camera
Cells
Embedded paraffin
Eosin
Formalin
Inflammation
Light microscopy
Microscope
Submandibular salivary glands
Corresponding Organization : University of Iowa
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Variable analysis
independent variables
- Harvesting and fixing of lacrimal and submandibular salivary glands in buffered formalin
- Processing, embedding in paraffin, and sectioning of the gland samples
- Inflammation, quantified using standard focus scoring
- Number of inflammatory foci per 4 mm^2 of the gland samples
- Staining of the gland sections with hematoxylin and eosin (H&E)
- Analysis of the gland sections using standard light microscopy
- Negative control: Not mentioned
- Positive control: Not mentioned
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