Tissuefax plus system

Bladder tissues were embedded in paraffin wax, cut into 3 µm sections, placed on slides, and stained with hematoxylin/eosin (H&E) by routine histopathological methods. Briefly, after deparaffinization and rehydration, the tissue sections were immersed in hematoxylin for 10 min and washed with tap water for 10 min. The slides were then immersed in eosin for 30 s and dehydrated by immersion in a series of increasing concentrations of alcohol. The sections were then immersed in xylene twice for 10 min each and coverslipped with mounting medium. The stained specimens were photographed using a TissueFAX Plus system (TissueGnostics, Vienna, Austria) and then analyzed histopathologically.

The TissueFAX Plus system (TISSUE GNOSTICS, Vienna, Austria) with the HistoQuest software 2.0 was employed for tissue staining analysis. Five 100 μm fields of view were examined per skin section. Dermal thickness was measured in five different regions of H&E-stained and Masson’s trichrome-stained skin slides to assess fibrosis. IHC staining quantified positively stained cells per square millimeter for TGF-β, α-SMA, collagen I, IL-6, IL-17A, IL-18, and TNF-α, using 100 μm views. Statistical comparisons were performed against a saline negative control group.