Fluoro-Jade C (FJC) was prepared from the powder form (AG325, MiliporeSigma, MO, USA) and used to detect degenerating neurons. Brain slices were incubated with 100% ethanol followed by 70% ethanol. Sections were then incubated in 0.06% potassium permanganate solution for 10 min. After washing with distilled water, sections were incubated in a 0.0001% solution of FJC dissolved in in 0.1% acetic acid for 10 min. Images were obtained using a Leica TCS SPE confocal system and a Leica DMi8 fluorescence microscope system using a FITC cube. Multiple images were captured with the 10X objective and stitched to generate images of the brain hemisphere (Leica LAS X software).
Dmi8 fluorescence microscope system
Manufactured by Leica
Sourced in United States
The Leica DMi8 is a fluorescence microscope system designed for advanced microscopic imaging. It features a high-performance illumination system, a sensitive camera, and comprehensive imaging software to capture and analyze fluorescent samples. The DMi8 is a versatile tool for researchers and scientists working in fields such as cell biology, neuroscience, and molecular biology.
Automatically generated - may contain errors
Lab products found in correlation
Las x software, by Leica (3 mentions)
Tcs spe confocal system, by Leica (3 mentions)
Ag325, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride, by Merck Group (1 mentions)
Rhodamine red conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Anti tom20 antibody, by Santa Cruz Biotechnology (1 mentions)
Rnascope multiplex fluorescent assay, by Advanced Cell Diagnostics (1 mentions)
Donkey anti rabbit igg alexa 594, by Thermo Fisher Scientific (1 mentions)
4 protocols using dmi8 fluorescence microscope system
1
Fluoro-Jade C Staining for Neurodegeneration
2
Immunofluorescence Staining of Cell Cultures
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Cells were fixed in 4% paraformaldehyde in PBS for 15 min, washed with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 7 min. After washing with PBS and blocking with 5% donkey serum for 20 min, cells were incubated with primary antibodies diluted in 1% donkey serum at room temperature for 1 h. The primary antibodies used in this study were anti-MAP2 (SantaCruz, sc-20172), anti-tubulin βIII (Sigma-Aldrich, MAB5564), anti-tyrosine hydroxylase (Santa Cruz, sc-25269), and anti-Tom20 antibody (Santa Cruz Biotech., sc-17764). After washing with PBS, samples were incubated with rhodamine red-conjugated anti-rabbit IgG (Jackson Immunoresearch, 111-295-003) diluted in PBS containing 1% donkey serum for 1 h at room temperature. For nuclear staining, cells were incubated with 1 μg/ml 4′,6′-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich) in PBS for 1 min at room temperature. Cells were washed three times with PBS and analyzed under a Leica DMi8 fluorescence microscope system.
3
Multiplex RNAscope Analysis of Ifi27l2a in Aged Mouse Brains
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The RNAscope fluorescent multiplex assay (Advanced Cell Diagnostics, Newark, CA, USA) was performed according to manufacturer’s instructions with 2 week post-stroke brains of aged mice (18–20 months) and aged sham to probe Ifi27l2a transcripts in brain cells. The murine Ifi27l2a probe was designed by ACD Biosystems, based on their own criteria. Brain sections (PFA fixed, 30-μm thickness) from the 2-week post stroke brain and sham brains of aged mice (18–20 month old) were hybridized with Ifi27l2a probes for 2 hours at 40°C. At the same time, ACD 3-plex positive control and negative control probes were incubated on one brain section to confirm signal specificity. The probes were amplified according to the manufacturer’s instructions and labeled with Opal-570 Red fluorophore (Akoya Biosciences, Marlborough, MA, USA). DAPI was used to label nuclei. Images were taken with a fluorescent microscope (Leica DMi8 fluorescence microscope system, Leica Biosystem, IL, USA) and a confocal microscope (Leica TCS SPE confocal system, Leica Biosystem, IL, USA). Multiple images were captured with the 10X objective covering the hemisphere and stitched to generate a single image (Leica LAS X software).
4
Immunostaining of IFITM3 in Mouse Brain
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Brains were harvested and then fixed for 24 h in 4% PFA at 4 °C. The brains were then transferred to 30% sucrose solution in PBS for an additional 24 h prior to generating 30 µm coronal sections with a frozen microtome (Micron HM 450, Thermo Fisher Scientific, Waltham, MA, USA). To begin immunostaining, sections were washed with PBS, incubated with blocking buffer (10% goat serum, 0.3% Trion X-100 in PBS), and incubated overnight at 4 °C with the following primary antibody: Rabbit anti-IFITM3 antibody (Thermo Fisher Scientific, Waltham, MA, USA). We used a donkey anti-rabbit IgG-Alexa 594 (1:200, Thermo Fisher Scientific, Waltham, MA, USA) as the secondary antibody. Sections were incubated with DAPI (4′,6-diamidino-2-phenylindole) to label nuclei. Images were obtained using a Leica TCS SPE confocal system and a Leica DMi8 fluorescence microscope system (Leica Biosystem, Richmond, IL, USA). Multiple images of the whole brain or individual hemispheres were captured with the 10× objective and stitched to generate a single high-resolution image (Leica LAS X software ver. 3.6.0.20104, Leica Biosystem, Nussloch, German). Image analysis was performed using ImageJ software (ver. 1.53q, National Institutes of Health, Bethesda, MD, USA).
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