Eosin y

Fresh frozen TNBC tissue sections were cryosectioned and thawed on ITO-coated glass slides. For hematoxylin and eosin (H&E) staining, the tissue sections underwent the following procedures: (1) air drying at room temperature for 15 min, (2) rinsing the slide in tap water for 5 min, (3) staining with Harris’ hematoxylin solution (Merck) for 3 min, (4) rinsing in tap water via quick dipping, (5) rinsing in 1% hydrochloric acid (HCl) in ethanol solution via quick dip, (6) rinsing in tap water for 5 min, and (7) staining with Eosin Y (BBC Biochemical) for 3 s. Afterward, the stained slides were washed and dehydrated using a series of ten dips in each of the following solutions: (1) water, (2) 70% ethanol, (3) 90% ethanol, and (4) 100% ethanol. A whole-slide image of the prepared slide was obtained using automated microscopy (Nikon Inverted Microscope ECLIPSE Ti-E).
For cell cluster isolation, H&E-stained tissue sections from two patients with TNBC (T1 and T2) were pathologically inspected to distinguish the spatial locations of cancer cells from normal cells. Based on the identified tumor cell locations, tumor cell clusters, each containing an average of 20 cells, were isolated from unstained fresh frozen tissue sections that were serial to the corresponding H&E sections.

For histological analysis, isolated femurs from euthanatized mice were fixed with 0.1 M phosphate buffer containing 4% paraformaldehyde (Sigma-Aldrich) at 4 °C for a 3 days. Then they were decalcified in 14.5% EDTA solution (sigma) for 1 weeks at room temperature. Decalcified tissues were rinsed, dehydrated, and paraffin embedded in accordance with general protocols. Paraffinized specimens were sectioned in 4 μm thickness with a microtome. Histological study was performed with Harris hematoxylin (BBC Biochemical, Vernon, WA, USA) and Eosin Y (BBC Biochemical). Images were obtained by inverted Nikon TS2R-FL microscope (Nikon).

All formalin-fixed, paraffin-embedded tissue blocks were sectioned into 5 μm-thick slices and stained with hematoxylin (Harris Hematoxylin; BBC Biochemical, WA, United States) and eosin (Eosin Y; BBC Biochemical).
When histopathological changes were identified in pancreatic tissue, including neutrophilic and lymphocytic inflammation, pancreatic necrosis, peripancreatic fat necrosis, edema, fibrosis, and atrophy, the sample was classified as having pancreatitis (21 (link)). In all tissue samples classified as having pancreatitis, the type of infiltrated inflammatory cells was recorded, and the semiquantitative histopathological grade was assessed based on the surface area affected by a lesion. When <10%, 10–40, and > 40% of the evaluated section were affected by a lesion, the sample was classified as having mild, moderate, and severe pancreatitis, respectively (21 (link)).
When the histopathological changes were absent or minimal changes were observed, the samples were defined as being normal pancreases.