For each animal, two coronal tissue sections that included the primary somatosensory cortex (S1) and CA1 field of the hippocampus (HPC) were imaged with a Nikon A1R HD confocal microscope using three excitation wavelengths: 488 (filter cube = 450/50), 561 (filter cube = 525/50), and 640 (filter cube = 595/50) nm simultaneously, using a 20x (Plan Apo VC 20x DIC N2, MRD70200) or 40x water immersion (Apo LWD 40x WI λS DIC N2, MRD77410) objective. All image analysis was carried out offline in NIH ImageJ (RRID:SCR_003070) or FIJI (RRID:SCR_002285) and performed masked to treatment by a single observer per experiment. For each type of analysis, one image per two sections per animal was analyzed. All image analysis protocols were generated in-house and results from identical or similar protocols have been previously published (Lowery et al., 2017 (link); Sipe et al., 2016 (link)).
A1r hd confocal microscope
Manufactured by Nikon
Sourced in United States, Japan
The Nikon A1R HD confocal microscope is a high-resolution imaging system designed for advanced research applications. It features a high-definition optical system and a resonant scanner for rapid image acquisition. The microscope is capable of capturing detailed images of biological samples at the cellular and subcellular levels.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements software, by Nikon (3 mentions)
Prolong glass antifade reagent, by Thermo Fisher Scientific (3 mentions)
Nis elements ar software, by Nikon (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
Prolong glass antifade, by Thermo Fisher Scientific (2 mentions)
Plan apo vc 20x dic n2, by Nikon (1 mentions)
Apo lwd 40x wi λs dic n2, by Nikon (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Phalloidin ifluor 555, by Abcam (1 mentions)
Air objective, by Nikon (1 mentions)
Nis elements ar analysis software, by Nikon (1 mentions)
Fluoro max dyed green aqueous fluorescent particles, by Thermo Fisher Scientific (1 mentions)
Rnascope multiplex fluorescent detection kit, by Advanced Cell Diagnostics (1 mentions)
Tunel kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Nucblue live readyprobe, by Thermo Fisher Scientific (1 mentions)
30 protocols using a1r hd confocal microscope
1
Multimodal Imaging of Cortical and Hippocampal Structures
2
Automated Analysis of RNAscope Data
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After the amplification procedure, slides were examined on a Nikon A1R HD confocal microscope (Nikon) using a 20 X objective. Images were first processed by removing background noise using the background subtraction tool (5.0-pixel rolling ball radius) in FIJI (Image J). Images from the same section were then registered using RNAscope Hiplex Image Registration Software v2. The signal was subsequently quantified with CellProfiler using a freely available pipeline (macros) for RNAscope (Patterson-Cross et al., 2021 (link)). A protocol with a step-by-step description of how to implement this pipeline for analyzing RNAscope data was recently published (Erben et al., 2018 (link); Erben and Buonanno, 2019 (link)). This pipeline was modified to include up to 12 mRNA probes and cells were considered positive for a given gene if they contained a minimum of seven mRNA transcripts (dots). Subsequently, all RNAscope data was analyzed by a blind experimenter. Sections from bregma –0.22 to –0.34 were considered anterior PVT and sections from bregma –1.58 to –1.70 were considered posterior PVT. Gene expression matrices were generated and analyzed using RStudio Version 4.2.0. Heatmaps and hierarchical clustering were performed using R functions pheatmap() and hclust().
3
Immunofluorescence Imaging of Fibroblast Proliferation
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Fixed human dermal fibroblasts (HDFs) cultured with and without Dex-MA solution were permeabilized with 0.1% triton X-100 in PBS at room temperature for 20 min, blocked with 5wt% goat serum in 0.01% triton X-100 at 4 °C overnight, and incubated with primary antibody Ki67 mouse monoclonal antibody (1:500, Abcam ab15580) in blocking buffer for 24 h at 4 °C. After 24 h, HDFs were incubated with goat anti-mouse Alexa Fluor 488 (1:1000, Life Technologies); counterstained for nuclei with Hoechst (1:500) and for actin cytoskeleton with phalloidin-Alexa Fluor 647 (1:1000, Thermo Fisher Scientific, Waltham, MA) in blocking buffer. Fluorescent images were acquired using a Nikon A1R HD confocal microscope. Unless otherwise specified, images are processed and presented as maximum intensity projections using Image J.
4
Cytotoxicity Evaluation of Dex-MA Hydrogels
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Human dermal fibroblasts (HDFs, passage 4–8) were cultured in DMEM media to 80% confluency on TCPS before seeding in 35 mm MatTek glass bottom dishes (100,000 cells per dish). After culturing for 24 h, cell media was replaced with 2 mL DMEM media containing 10 mg/mL 88% Dex-MA and live/dead cytotoxicity assay was performed after culturing HDFs in Dex-MA containing DMEM media for additional 24 h. Calcein AM (2 μM) plus Ethidium homodimer-1 (20 μM) were mixed in 10 mL PBS (pH = 7.4) to prepare the live/dead staining solution. Cultured cells were incubated with live/dead solution for 10–15 min at room temperature prior to imaging. Fluorescent images were acquired using a Nikon A1R HD confocal microscope. Unless otherwise specified, images were processed and presented as maximum intensity projections.
5
Imaging of L-Scaffolds and L-Hydrogels
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Repopulated L-Scaffolds and L-Hydrogels were fixed in 4% formaldehyde for 45 min at room temperature and stored in PBS with 0.05% sodium azide (Sigma-Aldrich) at 4 °C until further processing. This included permeabilization in 0.1% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and staining with 1X Phalloidin-iFluor 555 (Abcam, Cambridge, UK) for F-actin staining and DAPI (nuclei staining) prepared in 1% BSA in PBS for 1 h at room temperature. The L-Scaffolds and L-Hydrogels were immersed in Ce3D (prepared as previously described [21 (link)]) in chambers built on top of slides using iSpacers (SunJin Lab) and glass coverslips. They were imaged in a resonant scanner A1RHD confocal microscope (Nikon) controlled with the NIS Elements AR software (Nikon) using a 10× air objective (Nikon) and laser excitation. Images were corrected for brightness and prepared for publication in NIS Elements AR Analysis software (Nikon).
6
Quantitative Imaging of Cerebellar Vermis
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For each L7cre/Ai9+/−/Cx3cr1G/+ mouse, three sagittal sections including lobules IV/V of the cerebellar vermis were imaged at the URMC Center for Advanced Light Microscopy and Nanoscopy (CALMN) with a Nikon A1R HD confocal microscope with two excitation wavelengths: 488 (filter cube = 450/50) and 561(filter cube = 525/50) simultaneously, using a 10× (Plan Apo λD 10×, 0.45 NA, Nikon, Melville, NY, USA) or 40× water immersion (Apo LWD 40×/WI λS DIC N2, 1.15 NA, Nikon) objective. Imaging and analyses were performed blind to treatment and sex. Fixed-tissue image analysis was run with Ilastik, NIH ImageJ or FIJI, MATLAB, Cellpose ([36 (link)]; version 2.2.3; RRID:SCR_021716), Napari (napari.org; version 0.4.18; RRID:SCR_022765), and RStudio (posit.co; version 2023.09.1+494; RRID:SCR_000432).
7
Microgel Particle Characterization
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Microgel were prepared according to the aforementioned protocol.
100 nm Fluoro-Max Dyed Green Aqueous Fluorescent Particles (Thermo
Scientific Cat. no. G100) were diluted to a concentration of 1%, mixed
with microgel particles, and centrifuged at 1200g for 10 min. Confocal images were taken using a Nikon A1R HD confocal
microscope with a 10× objective (NA = 0.30) and 2.25 μm
z-stacks. Local thickness was calculated using an overlapping ball
algorithm (Local Thickness) built into FIJI, and mean and standard
deviations were calculated across each z-slice.
100 nm Fluoro-Max Dyed Green Aqueous Fluorescent Particles (Thermo
Scientific Cat. no. G100) were diluted to a concentration of 1%, mixed
with microgel particles, and centrifuged at 1200g for 10 min. Confocal images were taken using a Nikon A1R HD confocal
microscope with a 10× objective (NA = 0.30) and 2.25 μm
z-stacks. Local thickness was calculated using an overlapping ball
algorithm (Local Thickness) built into FIJI, and mean and standard
deviations were calculated across each z-slice.
8
Visualizing her1 and Venus transcripts in zebrafish PSM
9
TUNEL Assay for Apoptosis Detection
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A TUNEL kit (C10617, Life technologies) was used to detect apoptotic cells according to the manufacturer’s protocols. Samples were prepared per paraffin protocol outlined above and then incubated with TUNEL working solution for 1 h and shielded from light at 37 °C. The nuclei were stained by Dapi for 30 min. The specimens were imaged using a Nikon A1R HD confocal microscope with a DU4G filter-based detector, using a Super Plan Fluor LWD 20x 0.70NA air objective lens with digital zoom of 1x.
10
Cellular Uptake and Endosomal Escape of Luc mRNA LNPs
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HEK 293, HeLa, 4T1, and B16F10-Luc cells were seeded on glass slides overnight, followed by transfection with Luc mRNA@FITCLNPs for 0.5, 2, or 4 h. The fluorescence images were acquired with a Nikon A1R + HD Confocal Microscope. For the endosomal escape study, cells were seeded in confocal dishes at a density of 1 × 105 cells. Then, cells were transfected with LNPs encapsulating 0.5 μg/mL Luc Cy5mRNA. After 4 h, cells were stained with DAPI (ThermoFisher) and LysoBrite™ Green (AAT Bioquest), and the fluorescence signals of these cells were analyzed by CLSM.
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