Zen 2 pro software

At 0, 1, 4, 7, and 14 days post sciatic nerve crush, four rats from each group were intracardially perfused with 4% freshly depolymerized, neutral-buffered paraformaldehyde. Sciatic nerves were excised, immersed, and post-fixed in paraformaldehyde overnight at 4°C, and then dehydrated by 30% hypertonic sucrose solution for 48 hours. Following dehydration, tissues were embedded in Optimal Cutting Temperature compound and sectioned into 15-μm-thick slices on a cryostat microtome (CM3050, Leica Biosystems, Solms, Germany). Tissue slices with comparatively complete morphological structure were selected for subsequent observation. The slices were mounted on microscope slides, washed with phosphate-buffered saline, blocked with 5% goat serum for 30 minutes at room temperature, incubated with rabbit anti-total tau polyclonal antibody (1:200; Abcam) or rabbit anti-p-tau (Ser 404) polyclonal antibody (1:50; Thermo Fisher Scientific) overnight at 4°C, and then incubated with anti-rabbit Cy3 IgG (1:1,000; Sigma-Aldrich, St. Louis, MO, USA) for 2 hours at room temperature. Labeled sections were viewed with an optical and epifluorescence microscope (Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany), and images were captured and analyzed using Zen 2 Pro software (Zeiss).

Embryos mounted on 2% agar pads were anesthetized in 3 µl M9 buffer (1 liter contains 3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, and 1 mM MgSO₄) containing 33 mM sodium azide. The numbers of button-like cell corpses and pit-like vacuoles in the head region of living embryos at various developmental stages (comma, 1.5-fold, 2-fold, 2.5-fold, 3-fold, and 4-fold) were scored using DIC optics. 15 embryos at each developmental stage were scored for each strain. To examine embryonic cell corpse duration, embryos at the two-cell stage were mounted on 2% agar pads in egg salt buffer (118 mM NaCl and 48 mM KCl) at 20°C. Images in 30 Z-sections (1.0 µm/section) were captured every minute for 400 min using an Axioimager M2 microscope (ZEISS) equipped with an AxioCam monochrome digital camera (ZEISS). Images were processed and viewed using ZEN 2 pro software (ZEISS).