A1r hd laser scanning confocal microscope

Manufactured by Nikon

The A1R-HD laser-scanning confocal microscope is a high-resolution imaging system designed for advanced microscopy applications. It utilizes laser illumination and a specialized optical system to capture detailed, high-quality images of microscopic samples.

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Lab products found in correlation

Fluo 4 am, by Thermo Fisher Scientific (3 mentions) Cd31 bv421, by BioLegend (1 mentions) Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) Mom kit bmk 2202, by Vector Laboratories (1 mentions) Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) Superfrost plus slides, by Electron Microscopy Sciences (1 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)

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A1R laser scanning confocal microscope A1R laser scanning confocal A1R laser confocal microscope Laser scanning confocal microscope A1R HD confocal Nikon A1R HD microscope A1R confocal microscope Laser scanning microscope Laser confocal microscope A1R HD

6 protocols using a1r hd laser scanning confocal microscope

Whole Mount Microscopy of Tumor Lymphatics

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Whole mount microscopy was performed by excising tumors and lymph nodes from mice at endpoint. The afferent lymphatic vessel was dissected intact with the TDLN. Tissues were then placed on glass slides with two drops of PAB (1L PBS, 1g sodium azide, 10 g BSA). A coverslip was placed on top of the tissue and gently pressed down, excess PAB was removed by blotting. The tissue was then visualized via bright field and fluorescence microscopy of the same field of view. For confocal microscopy, tissues were prepared as above and the following antibodies were used for identification of blood and lymphatic vessels: CD31-BV421 (390, BioLegend) and LYVE-1-AF-488 (ALY7, eBioscience). Surface markers were stained for 30 minutes at 4°C in the dark, and washed two times with 2 mL of PAB for 2 minutes prior to mounting on slides. Images were captured using a Nikon A1R HD laser scanning confocal microscope using the high-speed resonant and galvano (non-resonant) scanner. All images were analyzed using ImageJ.

Han B.J., Murphy J.D., Qin S., Ye J., Uccello T.P., Garrett-Larsen J., Belt B.A., Prieto P.A., Egilmez N.K., Lord E.M., Linehan D.C., Mills B.N, & Gerber S.A. (2020). Microspheres Encapsulating Immunotherapy Agents Target the Tumor-Draining Lymph Node in Pancreatic Ductal Adenocarcinoma. Immunological investigations, 49(7), 808-823.

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Intracellular Calcium Dynamics in Ventricular Myocytes

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Ventricular myocytes were loaded with 8 μM Fluo-4 AM (Invitrogen, Thermo Fisher Scientific) for 25 minutes at room temperature, followed by 25 minutes of incubation in fresh external solution (deesterification) containing 140 mM NaCl, 5.4 mM KCl, 1.0 mM CaCl₂, 0.5 mM MgCl₂, 10 mM HEPES, and 5.6 mM glucose (pH 7.4, NaOH). Intracellular Ca²⁺ cycling was assessed using a Nikon A1R-HD laser-scanning confocal microscope with 488 nm excitation and 500–600 nm light collection. Myocytes were paced at 0.3 Hz using extracellular platinum electrodes. Only cells not exhibiting spontaneous Ca²⁺ oscillations and showing full recovery of Ca²⁺ transients in response to an electric stimuli were analyzed to obtain the Ca²⁺ wave frequency. Ca²⁺ sparks were recorded between stimuli and analyzed using Spark Master. To assess the SR Ca²⁺ load, 20 mM caffeine was applied at the end of the experiments. All experiments were performed at room temperature (26°C).

Tarasov M., Struckman H.L., Olgar Y., Miller A., Demirtas M., Bogdanov V., Terentyeva R., Soltisz A.M., Meng X., Min D., Sakuta G., Dunlap I., Duran A.D., Foster M.P., Davis J.P., Terentyev D., Györke S., Veeraraghavan R, & Radwański P.B. (2023). NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling. The Journal of Clinical Investigation, 133(7), e152071.

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Measuring PRMT5-Dependent Ca2+ Homeostasis

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Isolated activated (anti-CD3/CD28 – 2.5ug/ml, 50U IL-2, 48hr) CD4⁺ T cells from PRMT5^fl/fl and T-PRMT5^Δ/Δ mice were plated on poly-L lysine (Millipore Sigma, catalog no. P8920-100ML) coated glass-bottom dishes (Cellvis 35 mm - 14 mm micro-well #1.5 cover glass, Fisher Scientific, catalog no. NC0794151) for 120 min. Cells were then treated with 10 μmol Fluo-4-AM (Invitrogen, catalog no. F14201) dye for 30 min in DMEM (without phenol red and glutamine; catalog no. 11054020) at 5% CO₂ in a humidifying incubator at 37°C. Then the dye was washed out and cells were incubated for 30 min in modified EAE media supplemented with 10% FBS for de-esterification. Following de-esterification, cells were switched to modified Ringer’s solutions with 0 mM Ca²⁺ (120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 25 mM NaHCO₃, and 5.5 mM D-glucose, pH 7.3) for imaging with a Nikon A1R-HD laser-scanning confocal microscope. Fluo-4 was excited using 488 nm laser and fluorescence emission was detected at 500-550 nm. Resting Ca²⁺ baseline was recorded for 150 sec prior to addition of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (2μM). After 150 sec CaCl₂ (2mM) was added and calcium uptake was monitored for 600 sec. The data are represented as ΔF/F₀ vs. time, where F₀ is basal fluorescence and ΔF=F-F₀.

Sengupta S., West K.O., Sanghvi S., Laliotis G., Agosto L.M., Lynch K.W., Tsichlis P.N., Singh H., Patrick K.L, & Guerau-de-Arellano M. (2021). PRMT5 promotes symmetric dimethylation of RNA processing proteins and modulates activated T cell alternative splicing and Ca2+/NFAT signaling. ImmunoHorizons, 5(10), 884-897.

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PRMT5 Regulates Calcium Signaling

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Isolated activated (anti-CD3/CD28 -2.5ug/ml, 50U IL-2, 48hr) CD4 + T cells from PRMT5 fl/fl and T-PRMT5 Δ/Δ mice were plated on poly-L lysine (Millipore Sigma, catalog no. P8920-100ML) coated glassbottom dishes (Cellvis 35 mm -14 mm micro-well #1.5 cover glass, Fisher Scientific, catalog no. NC0794151) for 120 min. Cells were then treated with 10 μmol Fluo-4-AM (Invitrogen, catalog no. F14201) dye for 30 min in DMEM (without phenol red and glutamine; catalog no. 11054020) at 5% CO2 in a humidifying incubator at 37°C. Then the dye was washed out and cells were incubated for 30 min in modified EAE media supplemented with 10% FBS for de-esterification. Following deesterification, cells were switched to modified Ringer's solutions with 0 mM Ca 2+ (120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 25 mM NaHCO3, and 5.5 mM D-glucose, pH 7.3) for imaging with a Nikon A1R-HD laser-scanning confocal microscope. Fluo-4 was excited using 488 nm laser and fluorescence emission was detected at 500-550 nm. Resting Ca 2+ baseline was recorded for 150 sec prior to addition of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibitor thapsigargin (2μM). After 150 sec CaCl2 (2mM) was added and calcium uptake was monitored for 600 sec. The data are represented as ΔF/F0 vs. time, where F0 is basal fluorescence and ΔF=F-F0.

Sengupta S., West K.O., Sanghvi S., Laliotis G., Agosto L.M., Lynch K.W., Tsichlis P.N., Singh H., Patrick K.L., & Guerau‐de‐Arellano M. (2021). PRMT5 promotes symmetric dimethylation of RNA processing proteins and modulates activated T cell alternative splicing and Ca²⁺/NFAT signaling.

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Immunohistochemical Staining of Brain Slices

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Brain slices (30 μm) were prepared as previously described [19 (link)]. Sections were mounted on poly-D-lysine coated slides (Thermo Fisher), antigen retrieval with sodium citrate buffer was performed, except when staining with the TOC1 antibody, and the slides were blocked with PBS containing 5% BSA and 0.1% tween 20 for 1 h at room temperature. The sections were incubated with primary antibody in 5% BSA in PBS overnight at 4°C. The next day, slices were incubated for 1 h at room temperature with Alexa Fluor^™-conjugated secondary antibody including Alexa Fluor^™ 594 donkey-anti-rabbit, Alexa Fluor^™ 488 donkey-anti-rabbit, or Alexa Fluor^™ 647 donkey-anti-rabbit (Thermo Fisher Scientific). Alternatively, they were labeled using the MOM kit (BMK-2202, Vector laboratories), followed by three washes with PBS and labeling with Streptavidin Alexa Fluor^™ 488 or 647 (Thermo Fisher Scientific). The brain sections were coverslipped with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, P36961). The slides were imaged using a Nikon A1R HD laser scanning confocal microscope and recorded by NIS-Elements (Version 5.11) software. Resulting images were pseudocolored for illustration purposes.

Lin H., Sandkuhler S., Dunlea C., King D.H, & Johnson G.V. (2023). BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1. bioRxiv.

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Immunocytochemistry of Retinal Organoids

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For immunocytochemistry, organoids were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 40 min at room temperature, washed with PBS, cryopreserved in 30% sucrose, and sectioned on a cryostat. 15 μm sections were collected on Superfrost Plus slides (Electron Microscopy services), blocked for 1 hr at RT in 10% normal donkey serum, 5% BSA, and 0.5% Triton, then incubated overnight at 4°C with primary antibodies diluted in block. Primary antibodies, sources and dilutions are listed in key resources table. Slides were incubated with species-specific fluorophore-conjugated secondary antibodies diluted 1:500 in block, for 30 minutes in the dark at RT (donkey anti-mouse Alexa Fluor 488, donkey anti-rabbit AF546 and donkey anti goat-AF633: Thermo Fisher) and mounted with Prolong Gold antifade + DAPI to counterstain nuclei (Thermo Fisher). Sections were imaged on a Nikon A1R-HD laser scanning confocal microscope. For cone and rod counts, sections of the outer nuclear layer-like region of at least 4 individual organoids per line were immunostained with NR2E3 and cone ARR3, imaged, and rods and cones from at least 6 images were counted using Nikon Elements Analysis D software (Capowski et al., 2019 ; Kallman et al., 2020 (link)).

Saha A., Capowski E., Zepeda M.A., Nelson E.C., Gamm D.M, & Sinha R. (2022). Cone photoreceptors in human stem cell derived retinal organoids demonstrate intrinsic light responses that mimic those of primate fovea. Cell stem cell, 29(3), 460-471.e3.

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