For in vitro analyses, micro-TENNs were imaged using phase-contrast and fluorescence on a Nikon Eclipse Ti-S microscope with image acquisition using a QiClick camera interfaced with Nikon Elements. In order to determine the length of neurite penetration, the longest observable neurite in each micro-TENN was measured from the proximal end of the neuronal aggregate after fixation. For in vitro immunocytochemistry analyses, cultures and micro-TENNs were fluorescently imaged using a Nikon A1RSI Laser Scanning Confocal microscope. All micro-TENN confocal reconstructions were from full thickness z-stacks. For analysis of micro-TENNs post-transplant into the brain, micro-TENNs were fluorescently imaged using a Nikon A1RSI Laser Scanning Confocal microscope. Each section was analyzed to assess the presence, architecture, and outgrowth/integration of micro-TENN neurons/neurites.
A1rsi laser scanning confocal microscope
Manufactured by Nikon
Sourced in Japan, United States
The A1rsi laser scanning confocal microscope is a precision instrument designed for high-resolution imaging. It utilizes laser illumination and advanced optics to capture detailed, high-quality images of samples. The core function of this microscope is to provide researchers and analysts with a powerful tool for detailed observation and analysis of their specimens.
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Lab products found in correlation
Nis elements advanced research software, by Nikon (5 mentions)
Eclipse ti s microscope, by Nikon (3 mentions)
In situ apoptosis detection kit, by Takara Bio (3 mentions)
A1r laser scanning confocal microscope, by Nikon (2 mentions)
8 chamber cover glass bottom vessel, by Thermo Fisher Scientific (2 mentions)
Draq5, by Cell Signaling Technology (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Mzfliii, by Zeiss (1 mentions)
Anti αsma cy3, by Merck Group (1 mentions)
Qiclick camera, by Nikon (1 mentions)
Cm1850, by Leica (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 solution, by Thermo Fisher Scientific (1 mentions)
Cell tak cell and tissue adhesive, by Corning (1 mentions)
Nucblue fixed readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
8 chamber glass bottom vessel, by Thermo Fisher Scientific (1 mentions)
42 protocols using a1rsi laser scanning confocal microscope
1
Micro-TENN Microscopic Imaging and Analysis
2
Immunofluorescence Imaging of Cancer Cells
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The cancer cell lines were grown to about 80% confluency in 8-well confocal imaging chamber plates. The cells were incubated with 10 nM antibodies at different time points. The cells were washed twice with PBS and incubated with propidium iodide at room temperature in the dark for 5 min. Then the cells were stained with Hoechst 33342 (Cat No: H1399, Life TechnologiesTM.) for 5 min. Cells were then washed three times with PBS (pH 7.4) and used for confocal imaging. Confocal fluorescence images of cells were obtained using a Nikon A1R-si Laser Scanning Confocal Microscope (Japan), equipped with lasers of 405/488/561/638 nm.
3
Immunostaining and Imaging of Adult Gut and Wing Discs
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Adult guts were dissected in PBS and fixed in 4% paraformaldehyde for 45 mins at room temperature; wing discs from 3rd instar larvae were fixed in 4% paraformaldehyde for 20 mins at room temperature. Tissues were washed with PBS + 0.1% Triton X-100 and blocked with PBS + 0.1% Tween-20 + 10% BSA for 1 h at room temperature. The samples were incubated with primary antibody (diluted in PBS + 0.5% Triton X-100) at 4 °C for 1–3 days. Secondary antibody incubation was carried out at room temperature for 2 h. The samples were subsequently stained with DAPI (2 μg/ml) and mounted in Prolong Gold Antifade Reagent (Invitrogen). Confocal images were captured on a Nikon A1RSi laser scanning confocal microscope, Nikon CSU-W1 spinning disk confocal microscope, or Nikon Yokogawa CSU-W1 SoRa spinning disk confocal microscope and processed with Adobe Photoshop / Illustrator software from Adobe Suite 2023. Adult wings were mounted in Mowiol and their images acquired using a Leica MZFLIII stereomicroscope with a Zeiss Axiocam 208 camera and Nikon Zen 3.0 software.
4
Histological Characterization of Wound Tissue
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To measure histological characteristics of wounds, each wound was embedded and sectioned through its entirety. For histology analysis, 10 μm cryosections from each wound tissue were fixed in 4% formaldehyde and stained with hematoxylin and eosin. For immunofluorescence analysis, 10 μm cryosections were fixed in ice-cold acetone for 10 minutes and blocked for 1 hour at room temperature. Sections were incubated with anti-α-SMA-Cy3 (Sigma C6198) antibody at 4°C, overnight. After 5 min counterstaining with 4′, 6-diamidino-2-phenylindole (DAPI), the slides were mounted and analyzed using Nikon A1RSi Laser Scanning Confocal Microscope.
5
Neurite Characterization in 3D Tissue-Engineered Constructs
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For in vitro analyses, TE-NSPs were imaged using phase contrast and fluorescence on a Nikon Eclipse Ti-S microscope with image acquisition using a QiClick camera interfaced with Nikon Elements. In order to determine the length of neurite penetration, the longest observable neurite in each TE-NSP was measured from the proximal end of the neuronal aggregate after fixation. For in vitro immunocytochemistry analyses, cultures and TE-NSPs were fluorescently imaged using a Nikon A1RSI Laser Scanning Confocal microscope. All TE-NSP confocal reconstructions were from full-thickness z-stacks. In order to determine dopaminergic purity, the number of TH+ neurons was divided by the number of β-tubulin III+ neurons at 14 DIV.
6
Quantitative Dendritic Spine Analysis
7
Spectral Separation of Low-intensity EGFP
8
Dual Labeling for Apoptotic Myofibroblasts
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Double immunofluorescence staining for TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) and α-SMA was performed to examine apoptotic myofibroblasts in granulation tissues. α-SMA staining was initially performed in cryosections according to the protocol described above. Then TUNEL staining was performed using an In Situ Apoptosis Detection Kit (Takara, MK500). After nuclear DNA was probed with DAPI, the slides were mounted and analyzed by Nikon A1RSi Laser Scanning Confocal Microscope. Negative control was obtained by omitting terminal deoxynucleotidyl transferase (TdT) enzyme from the labeling procedure.
9
Autophagosome Formation Quantification
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About 1.4 × 105 cells were plated into each well of a 24-well plate 24 h before transfection. Plasmids (800 ng/well) or siRNA (660 ng/well) were transfected with Lipofectamine 2000 according to the manufacturer’s protocol. Both fixed and live cells were imaged by using an A1R-si Laser Scanning Confocal Microscope (Nikon, Japan). To measure the formation of autophagosomes, HeLa cells were transfected with plasmids encoding EGFP-LC3. The accumulation and distribution of EGFP-LC3 puncta were recorded using an A1R-si Laser Scanning Confocal Microscope. Ten fields of cells with EGFP-LC3 puncta were counted. The number of EGFP-LC3 puncta per cell was counted for 50 cells.
10
Analyzing TE-NSPs in Post-Transplant
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For the analysis of TE-NSPs post-transplant, TE-NSPs were fluorescently imaged using either a Nikon A1RSI Laser Scanning Confocal microscope or a Nikon A1R HD Multiphoton microscope. A skilled researcher blinded to the enrollment group made all assessments. Each section was analyzed to assess the presence, architecture, and outgrowth/integration of TE-NSP neurons/neurites. Within the histologically assessed samples, the health of the implanted aggregates or constructs was described as exhibiting a clear presence of cell survival or no evidence of cell survival. Implanted aggregates or constructs were also classified based on presence or absence of axonal tracts spanning the length of the micro-column. These binary outcome metrics were utilized to determine the frequency of cell survival or axonal growth across implant conditions at early and later time points.
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