Cytopainter phalloidin ifluor 555

Sections of snap-frozen liver were cut at 5 microns using a cryomicrotome and stained with hematoxylin and eosin. Formalin-fixed sections of liver were processed routinely into paraffin blocks, sectioned at 5 microns, and stained with hematoxylin and eosin. Additional sections were used for immunohistochemical (IHC) and immunofluorescence (IF) staining. Antibodies directed against myeloperoxidase (ab9535, Abcam, Cambridge, MA) to detect neutrophils and cleaved caspase-3 (CP2298, Biocare Medical, Pacheco, CA) to detect apoptotic cells were used followed by secondary reagents, including Bond Polymer Refine Detection system (DS9800, Leica, Buffalo Grove, IL) for chromogenic IHC and goat anti-rabbit Alexa Fluor 488 (ab150077, Abcam) for immunofluorescence staining. For IF, polymerized actin of the cytoskeleton was detected using phalloidin (Cytopainter Phalloidin-iFluor 555, ab176756, Abcam) and nuclei were detected using DAPI (Fluoroshield Mounting Medium with DAPI, ab104139, Abcam). Stained sections were visualized using a Leica DM2500 microscope with a Leica DFC495 camera and Leica Application Suite v4.12 software or an Olympus BX41 with an Olympus U-RFL-T source (Olympus, Center Valley, PA).

In addition to the imaged tissue section, additional liver sections were obtained as described above (Liver procurement and treatment) and a single dose of TFA (100 μL) in one of four concentrations (0.25, 0.5, 1.0, or 2.0M) was injected into eight additional tissue sections (two sections per tested concentration). Liver sections were bisected and a portion was fixed in 10% neutral buffered formalin. Samples were processed routinely, embedded in paraffin, sectioned at 4-μm thickness, stained with hematoxylin and eosin, and examined by a board-certified veterinary pathologist. Phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) staining were also used to highlight cytoskeletal and nuclear structures, respectively (CytoPainter Phalloidin-iFluor 555 [ab176756] and Fluoroshield Mounting Medium with DAPI [ab104139] from Abcam plc, Cambridge, UK).