Gemini stainer

BPSEs were fixed in 10% formalin overnight at 4°C, then stored in 70% ethanol until ready for processing. Human postmortem samples were obtained from Tissue4research.com. Tissues were processed using the standard processing procedure on the ThermoFisher Excelsior AS Tissue Processor. Tissues were embedded in paraffin wax using the ThermoFisher HistoStar Embedding Workstation. Six-micrometer sections were cut on the ThermoFisher HM 355S Microtome and placed on positively charged slides. H&E staining was performed on the ThermoFisher Gemini stainer using the predefined H&E protocol. IHC was performed on the Leica Bond RXm using the predefined “Standard Protocol F” and the Bond Polymer Refine Detection Kit for DAB staining (Leica DS9800) with the following primary antibodies: COL-1 (Invitrogen PA126204), COL-VII (abcam ab6312), Ki67 (Leica PA0118), CK-1 (BioLegend 905201), FLG01 (Invitrogen MA5-13440), CK-15 (abcam ab52816), ZO-1 (Invitrogen 61-7300), CLD1 (abcam ab211737), and CDH1 (abcam ab1416). Slide images were taken with the Leica Aperio Versa 200.

For histological and immunohistochemistry analyses, mice were euthanized by carbon dioxide asphyxiation. Lymphoid tissues were harvested, fixed overnight with 10% neutral buffered formalin (VWR), transferred to 70% ethanol solution, and subsequently embedded in parafilm. Sections were cut at a thickness of 10 µm and stained with hematoxylin and eosin (H&E) for pathological assessment. Immunohistochemistry was performed on a Thermo Autostainer 360 machine. Slides were antigen-retrieved using Thermo citrate buffer (pH 6.0) in the pretreatment module. Sections were treated with Biocare rodent block, primary antibody, and anti-rabbit HRP-polymer (Vector Laboratories). The slides were developed with Thermo Ultra DAB and counterstained with hematoxylin in a Thermo Gemini stainer, and coverslips were added using the Thermo Consul coverslipper.