Harris hematoxylin solution

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, all animals were deeply anesthetized and transcardially perfused with 150 mL of saline, followed by 500 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.2) via a peristaltic pump. After perfusion, the spinal cord was removed, postfixed in 4% PFA overnight, and cryoprotected in 30% sucrose for 3 days. The tissues were embedded in M1 compound (Thermo Fisher Scientific Inc., Waltham, MA, USA) and sectioned sagittally at a thickness of 16 µm on a cryostat. H&E staining was performed at each time point after SCI. Sections were washed in 0.1 M PBS and immersed in Harris hematoxylin solution (Merck KGaA, Darmstadt, Germany) for 3 min followed by a brief wash in distilled water. Slides were then immersed briefly in 1% acid alcohol (1% HCl in 70% ethanol) and stained with eosin Y solution (BBC biochemical, Mount Vernon, WA, USA) for 30 s. The slides were dehydrated with an ethanol series, cleared with xylene, mounted in DPX (Merck KGaA, Darmstadt, Germany), and observed under a microscope (Nikon, Tokyo, Japan).

Tween 80, DMSO (dimethyl sulfoxide), normal saline (0.9% NaCl), formalin, phosphate buffer solution (PBS), pentylenetetrazole (PTZ), diazepam, NFkB ELISA kit (Santa Cruz Biotechnology, Santa Cruz, CA, United States) xylene, ethyl alcohol,% HCL, 1% ammonia, Harris’ hematoxylin solution (Sigma-Aldrich, St. Louis, MO, United States) and eosin Y, bicinchoninic acid (BCA) protein test kit, 12% SDS gel.