Q color5 imaging system

5 μM sections of the proximal aorta were fixed in cold acetone and incubated at room temperature in 2% BSA/PBS. Slides were treated with avidin block (Vector, Olean, NY), biotin block (Vector), and peroxidase-activity block (9:1 ratio of methanol:30% H₂O₂). Macrophages were stained using a 1:25 dilution of rat-anti mouse macrophage/monocyte, clone MOMA-2 (MilliporeSigma) in 2% BSA/PBS for 1 hour at RT. Next, slides were stained with a 1:200 dilution of biotin goat-anti rat (BD Biosciences) in 2% BSA/PBS for 30 minutes at 37°C.
Strepavadin-HRP (Biogenex, Fremont, CA) was applied for 20 minutes followed by AEC substrate (Abcam) for 2 minutes. Hematoxylin counterstain was applied for 2 minutes and slides were imaged using a Q-Color5™ imaging system (Olympus, Center Valley, PA). Quantification of macrophage/monocyte area was performed using ImageJ and averaged for four sections.

Explanted gingival tissues from A20^+/+ (n=6) and A20^+/− (n=6) mice (males, littermates) were fixed in 4% formaldehyde, sectioned and stained with hematoxylin and eosin (H&E) following standard protocol. Histology images were acquired using QColor 5 Imaging System from Olympus Microscopy with a 10X magnification objective lens. Quantification of numbers of nucleated cells (hematoxylin-positive) in the gingival was performed by a blinded examiner using CellSens software at 20X magnification. Counting was performed using the ‘Count and Measure’ tool and the ‘Manual Threshold’ option to choose an initial nucleated cell for reference. Once the original cell was selected, subsequent cells were automatically selected, until all nucleated cells were highlighted. Data were reported as area stained (μm²) in each field of view. Nine regions/fields of view were analyzed per ligated side, and six regions/fields of view were analyzed per control side, per mouse respectively.