Sz61 dissecting microscope

The mice were anesthetized with ketamine hydrochloride (100 mg/kg, intramuscular) and 2% xylazine hydrochloride (10 mg/kg, Abbott Laboratories, USA). The auditory bullae was rapidly removed and placed in a Petri dish filled with cold, fresh artificial perilymph solution. The surgery was performed under sterile conditions. The vestibular sacculus, utriculus, and ampullae of the semicircular canal tissue were gently extracted and placed in a cold artificial perilymph solution containing 125 mmol/L NaCl, 3.5 mmol/L KCl, 1.3 mmol/L CaCl₂, 1.5 mmol/L MgCl₂, 0.51 mmol/L NaH₂PO4, 10 mmol/L Hepes, and 5 mmol/L glucose at pH 7.4 with osmolarity adjusted to 310 mmol/kg. Otoliths were gently removed with Dumont tweezers (110 mm, 0.1*0.06 mm tip) and a Tungsten dissecting probe (50 mm, 0.5 mm diameter rod). The tissue was torn into pieces as small as possible under sterile conditions with fine ophthalmic tweezers under an Olympus SZ61 dissecting microscope using the method described by Neng et al. (Neng et al., 2013b (link)). After the tiny fragments of tissue resettled by gravity, they were repositioned to uniform density with a needle (50 mm, 0.5 mm diameter rod). The tiny fragments were then selectively cultured in one of three specific growth media, each specifically formulated to support the growth of either ECs, PCs, or PVM/Ms.

To determine alveolar bone loss, the maxillae were hemisected, mechanically defleshed and exposed overnight to 3% hydrogen peroxide. They were then treated for 5 min with 1% sodium hypochlorite and then stained with methylene blue dye (Fisher Scientific Company, Fair Lawn, New Jersey) in order to delineate the cemento-enamel junction. The jaws were mounted in utility wax, and the lingual surfaces of the molars were photographed with 10X magnification using an Olympus (SZ61) dissecting microscope (Olympus, Center Valley, PA). The images were digitalized [33 (link)] and printed on A4 paper, and bone loss around the three molars was measured at 12 sites by two different examiners. Results were presented in millimeters, and the significance in bone loss was calculated by comparing bone loss means of the control group with those of the infected diabetic and non-diabetic group of WT and LFKO^−/− mice using one-way analysis of variance (ANOVA) [38 (link)].