To examine the formation of AS2 bodies, we established three to five lines of transgenic Arabidopsis plants for analysis of the subcellular localization of AS2‐YFP and as2‐variant‐YFPs. Patterns of fluorescence due to YFP and 4,6‐diamidino‐2‐phenylindole (DAPI) were analyzed in 4 to 75 cells of each line of transgenic plants after induction of expression of transgenes. For DAPI staining, Arabidopsis cotyledons and shoot apices containing leaf primordia were separated, incubated in 3.7% paraformaldehyde for 30 min and stained with 0.2 µg/ml DAPI. For DAPI staining of cultured MM2d and BY‐2 cells, cells were fixed in 3.7% paraformaldehyde in phosphate‐buffered saline (PBS; pH 7.2) for 14 min and stained with 0.2 µg/ml DAPI. Images were recorded by confocal laser scanning fluorescence microscopy with a ×40 magnification 1.3 NA plan apochromat oil immersion objective (FV1000 confocal laser scanning fluorescence microscope; OLYMPUS, Tokyo, Japan).
Fv1000 laser scanning confocal fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The FV1000 is a laser scanning confocal fluorescence microscope designed for high-resolution imaging of biological samples. It utilizes a laser source to excite fluorescent probes within the sample, and a photomultiplier tube detects the emitted fluorescence. The microscope scans the sample in a raster pattern, building a digital image from the detected fluorescence signals. The FV1000 provides optical sectioning capabilities, allowing for the capture of three-dimensional data from the specimen.
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Lab products found in correlation
Tris buffered saline (tbs), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Pbs 1x, by Thermo Fisher Scientific (2 mentions)
Shandon cryotome e cryostat, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
6 well plate, by Corning (1 mentions)
Tandem gfp rfp lc3 adenovirus, by Hanbio Biotechnology (1 mentions)
5 protocols using fv1000 laser scanning confocal fluorescence microscope
1
Visualizing AS2 Bodies in Arabidopsis
2
Subcellular Localization of IPN2
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Stereo fluorescence microscope was used for light microscopy and fluorescence detection. For subcellular localization of IPN2 in root nodules, IPN2 antibody was used for in situ immunolocalization according to the method described (Wang et al., 2015) . Olympus FV1000 confocal laser-scanning fluorescence microscope was used for confocal imaging. Fluorescence was excited at 488 nm for YFP and 559 nm for mCherry. Emission wave lengths were detected at 505-550 nm for YFP and 585-615 nm for mCherry.
3
Tracing Autophagy with GFP-RFP-LC3 Fusion
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GFP-RFP-LC3 fusion protein (two-color fluorescent) was used to trace the process of autophagy. RFP-GFP-LC3 emitted both green and red fluorescence signals when the protein localizes to autophagosomes (yellow signals in merged pictures) in autophagy induction process. Renal cancer cells were seeded onto glass cover slides (1 × 1.5 cm) in 6-well plates (Corning) overnight. The cells were transfected with tandem GFP-RFP-LC3 adenovirus (Hanbio, Shanghai, China) according to the manufacturer’s instruction. Cells were incubated for 24 h, washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI, and coverslipped. Confocal images were obtained using an Olympus FV1000 Laser Scanning confocal fluorescence microscope (Tokyo, Japan). Autophagosomes displaying both green and red fluorescence were evaluated by LC3 puncta number per cell quantified using ImageJ software.
4
Quantifying Microglia and Vascular Changes
5
Microglia and Endothelial Cell Quantification
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Following seven days upon the treatment, mice were weighed and deeply anesthetized with a ketamine/xylazine cocktail accordingly. Animals were transcardially perfused once with ice-cold PBS 1x (Gibco) and then with 4% paraformaldehyde (PFA). Brains were collected in 4% PFA for an additional 24 h postfixation and incubated in 20% sucrose for another 24 h.
For immunofluorescence assay, brains were embedded in Tissue-Tek ® optimal cutting temperature compound (Sakura), cut into 10 μm sections of thickness using a Shandon Cryotome E cryostat (Thermo Scientific), and mounted on Starfrost ® adhesive slides (Knittel Glass). Sections were rehydrated with blocking buffer (10% BSA, 0.3% Triton in TBS), rinsed with TBS (Gibco), and incubated overnight at 4°C with the corresponding dilutions of the antibodies CD45 (BioLegend, clone 30-F11) and CD31 (Santa Cruz, clone M-20) in blocking buffer. After several rinses, sections were incubated with Alexa Fluor 488 (Molecular Probes) or Alexa Fluor 546 (Molecular Probes) antibodies and counterstained with DAPI. Slides were analyzed under a FV1000 laser scanning confocal fluorescence microscope (Olympus). Quantification of microglia numbers and CD45 integrated density area was performed using ImageJ software (NIH, USA).
For immunofluorescence assay, brains were embedded in Tissue-Tek ® optimal cutting temperature compound (Sakura), cut into 10 μm sections of thickness using a Shandon Cryotome E cryostat (Thermo Scientific), and mounted on Starfrost ® adhesive slides (Knittel Glass). Sections were rehydrated with blocking buffer (10% BSA, 0.3% Triton in TBS), rinsed with TBS (Gibco), and incubated overnight at 4°C with the corresponding dilutions of the antibodies CD45 (BioLegend, clone 30-F11) and CD31 (Santa Cruz, clone M-20) in blocking buffer. After several rinses, sections were incubated with Alexa Fluor 488 (Molecular Probes) or Alexa Fluor 546 (Molecular Probes) antibodies and counterstained with DAPI. Slides were analyzed under a FV1000 laser scanning confocal fluorescence microscope (Olympus). Quantification of microglia numbers and CD45 integrated density area was performed using ImageJ software (NIH, USA).
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