After the behavior tests, the brain slices for immunohistochemistry staining were prepared from the intracardially perfused brains, and staining procedures were used as described previously (Sun et al. 2018 (link)). All frozen brain sections were washed with 0.3% Triton X-100 PBS and then blocked (10% goat serum, 0.1% Triton X-100 in PBS) for 2 h at 4 °C. Slices were incubated with mouse goat anti-Iba-1 (1:1000) in blocking solution for 24 h at 4 °C. Next, the sections were rinsed with PBS and incubated with donkey anti-goat IgG Alexa Fluor 594 (1:200) in PBS for 1 h at room temperature. All antibodies were diluted in PBS with 0.1% Triton X-100 and 2% bovine serum albumin. Nuclei were counter-stained with Hoechst33258 (1:100). The brain slices were then moved to slides, coverslipped with 50% glycerin, and photographed with a FluoView FV100 microscope (Olympus).
Fluoview fv100 microscope
Manufactured by Olympus
Sourced in Japan
The Fluoview FV100 is a high-performance confocal microscope system designed for advanced fluorescence imaging. It features high-resolution optics, sensitive detection, and a modular design to accommodate a wide range of sample types and applications.
Automatically generated - may contain errors
4 protocols using fluoview fv100 microscope
1
Immunohistochemistry Staining of Brain Slices
2
Micronucleation Assay Protocol
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Micronucleation experiments were performed as previously described (14 (link)). Briefly, cells were treated with 680C91 (20 μM) or KYN (60 μM) 24 h prior to BCNU treatment. Cells were treated with BCNU (125 μM) for 24 h before treating with cytochalasin-B (2 μg/ml) (Sigma Aldrich; Cat# 250233, St. Louis, MO) for an additional 24 h. Slides were stained with α-tubulin. Detailed antibody information can be found in Supplementary Table S1 . Images were obtained via Olympus Fluoview FV100 microscope and subjected to blinded scoring in biological triplicate.
3
Immunohistochemical Analysis of Microglia in Mouse ACC
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After behavior tests, mice were anesthetized with diethyl ether and perfused with 4% polyformaldehyde. The brain was isolated from the mice and was immersed in 4% paraformaldehyde overnight and then dehydrated through an ascending sucrose series (15% and 30% (w/v) sucrose in 0.1 M PBS) at 4°C overnight respectively. Coronal sections (30 μm) from the ACC were cut by a cryogenic constant sectioning device (Leica, CM1950), immediately mounted on superfrost slides, and stored at 4°C overnight to dry. The next day, all sections were washed with 0.3% Triton X-100 PBS and then were blocked (10% goat serum, 0.1% Triton X-100 in PBS) for 2 hours at 4°C. The brain sections were incubated with goat-anti-Iba-1(1:100; Abcam, ab178847) in blocking solution for 16 h at 4°C and washed with PBS, further incubated with goat anti-rabbit IgG Alexa Fluor 594 (1:200; Abcam, ab150080) in PBS for 1.5 h at room temperature in the dark environment. Nuclei were counter-stained with Hoechst 33258 (Sigma, 94403). The slides were coverslipped with 50% glycerine and were photographed with an Olympus Fluoview FV100 microscope (Olympus, Japan).
4
Quantifying Myocardial Apoptosis via TUNEL
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The degree of myocardial and cellular apoptosis was measured by TUNEL analysis using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer's instructions. Nuclei were visualized by DAPI staining. The samples were examined under an Olympus Fluoview FV100 microscope (Olympus, Tokyo, Japan), and the results are presented as an apoptotic index (×100%).
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