Anti gfp

Animals were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg) and intracardially perfused with 30 ml of PBS followed by a fixative containing 4% paraformaldehyde in 0.1 M PB. After perfusion fixation, animal tissues were kept in the same fixative at 4°C for 18 h and permeated with 15% (w/v) sucrose buffer with agitation. Each tissue was embedded in Tissue-Tek O.C.T compound (Sakura, Tokyo, Japan), frozen with liquid nitrogen and cut on a cryostat at 8 μm sections collected on MAS (Matsunami aggressive silane)-coated glass slides. Sections were blocked with 3% normal goat serum in PBS at room temperature for 15 min and processed for immunohistochemistry. The following antibodies were used: anti-TNF-α (1∶100, Abcam, Cambridge, United Kingdom), anti-GFP (1∶100, Medical & Biological Laboratories, Nagoya, Japan), anti-cleaved caspase3 (1∶100, Cell Signaling Technology Japan, Tokyo, Japan), and AlexaFluor 488, 594 and 633 (1∶1000, Abcam) as primary and secondary antibodies. For marker stains, we used 4′,6-diamidino-2-phenylindole (DAPI) for nuclei and fluorescent Nissl (NeuroTrace 435/455 or 530/615, Molecular Probes, Eugene, Oregon, USA) for neurons. Fluorescence images were captured and analyzed using Nikon C1si and the software, EZ-C1 version 3.90 (Nikon, Tokyo Japan).

The YOM38 yeast cell containing the pR316-GFP-Atg8 plasmid yeast strain in glucose liquid medium with initial OD₆₀₀ value of 0.1 was treated with 0, 0.3, 1, and 3 µM GENI or 300 µM RES for 22 h. The yeast cells of different groups were collected and sonicated for 5 min. Cell lysates were centrifuged at 12,000× g for 15 min to obtain the supernatants for Western blot. Protein concentrations were measured using a BCA protein assay kit (CoWin Biotech, Beijing, China). Western blot was performed as described in our previous study [20 (link)]. Approximately 20 µg protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto poly (vinylidene fluoride) membranes. Membranes were incubated with primary antibodies followed by secondary antibodies. The primary antibodies used were as follows: anti-GFP (Medical & Biological Laboratories, Nagoya, Japan) and anti-β-actin (CoWin Biotech, Beijing, China). The secondary antibodies used were horseradish peroxidase-linked antirabbit IgGs (CoWin Biotech, Beijing, China). Antigens were visualized using an eECL Western Blot Kit (CoWin Biotech, Beijing, China) and digitized using the ImageJ software (National Institute of Health, Rockville, MD, USA).