Asp300

Right kidney sample tissues fixed in 10% neutral formalin were managed in a tissue processing machine (ASP300s, Leica Biosystems, IL, USA); each sample was embedded in paraffin wax and sliced into 4–5-µm-thick sections. Three sections of each group were chosen and stained with hematoxylin and eosin (H&E) dye, periodic acid–Schiff (PAS) stain, and Masson’s trichrome (MT) stain [57 (link)]. All the sections of tissue were analyzed using a microscope (Olympus BX 52) for histopathological description which was recorded by a histopathologist, blinded to the experimental groups. Olympus DP21 camera fixed over the microscope was used for taking photographs.

The right maxilla was resected from each mouse and immediately fixed in 4% paraformaldehyde for 24 h, then decalcified with diethyl pyrocarbonate-treated 0.5 M ethylenediaminetetraacetic acid (pH 8) for 28 days at room temperature. The decalcified specimens were then dehydrated and embedded in paraffin using a fully-enclosed tissue processor (ASP300S, Leica Biosystems, Buffalo Grove, IL, USA). Tissue blocks were cut into serial sections (4 μm) parallel to the mesiodistal plane using a microtome, then sections were stained with Mayer’s hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) and eosin Y solution (Sigma-Aldrich) for assessment of inflammation. The sections were examined with a stereomicroscope.
The number of inflammatory cells (round-shaped nuclei) and gingival fibroblast (spindle-shaped nuclei) within a square field (100 × 100 μm) in connective tissue adjacent to the gingival epithelium between first and second molars were morphologically evaluated and counted in three tissue sections per mouse specimen (n = 3 per group). Similarly, the number of periodontal ligament (PDL) cells (spindle-shaped nuclei in the PDL space) and alveolar bone lining cells (cell nuclei on bone surface) were counted. All cell counts were averaged for each group, and data were expressed as the mean number of cells per 1.0 mm² of connective tissue in the maxillary specimens.

One half of bladder tissues from UTI infected and excipient control mice were collected for histopathological analysis. Gross observations were made, and subsequently tissues were fixed in 10% neutral buffered formalin for 48–72 h. Tissues were processed through graded alcohols, xylene and paraffin on an ASP300S tissue processor (Leica Biosystems, Buffalo Grove, IL, USA) and embedded into paraffin blocks. Then, 4 µm tissue sections were cut, mounted on glass slides, and stained with hematoxylin and eosin (H&E) (SelecTech; Leica Biosystems). The slides were examined by a veterinary pathologist.