Ds ri2 microscope

A small part of the liver tissue was fixed with 10% neutral buffer formalin solution for 24 h, and then dehydrated by different grades of ethanol (80%, 95% and 100%), transparent in xylene and covered with paraffin. A 0.2 μm slice was then cut from paraffin-embedded tissue using a slicer (Leica Instruments GmbH, Wetzlar, Germany). The slides were prepared by dewaxing, stained with H&E, and sealed with neutral gum. Microphotographs were obtained using a Nikon DS-Ri2 microscope (Nikon Corporation, Tokyo, Japan).
Another part of the liver tissue was fixed with 2.5% glutaraldehyde and 4% paraformaldehyde at 4 °C for 2 h, rinsed with 0.1 M phosphate buffer, and then fixed with 1% osmic acid at 4 °C for 2 h. After rinsing with 0.1 M phosphate buffer, different grades of acetone (30%, 50%, 70%, 90% and 100%) were dehydrated. After embedding, sections were stained with uranium acetate and lead citrate, and finally observed using a JEM-1400 PLUS transmission electron microscope (Japan Electronics Corporation, Tokyo, Japan).

Immunofluorescence staining was performed as previously described.³⁴ Briefly, frozen brain sections (8‐μm thickness) were made and then permeabilized and blocked in 5% donkey serum consisting of 0.3% Triton X‐100 for 1 h at room temperature. Primary antibodies were diluted by blocking buffer and incubated with sections for 16 h at 4°C. After being incubated with secondary antibodies, nuclei were stained by DAPI (4,6‐diamidino‐2‐phenylindole) and observed by Nikon DS‐Ri2 microscope (Nikon).

TUNEL analysis was performed using an In Situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland) according to manufacturer's instructions. Briefly, brain sections were hydrated via deionized water and then permeabilized by 0.3% Triton X‐100 in TBS. TdT and fluorescein‐labeled dUTP were mixed to form the TUNEL staining solution, which was then incubated with brain slides for 20 min at 37°C in the dark. The results were observed via Nikon DS‐Ri2 microscope (Nikon, Tokyo, Japan).