Deltavision ultra microscope

Manufactured by GE Healthcare

Sourced in United States

The DeltaVision Ultra microscope is a high-performance imaging system designed for advanced microscopy applications. It features a powerful optical system and state-of-the-art technology to provide superior image quality and resolution. The core function of the DeltaVision Ultra is to enable detailed visualization and analysis of samples at the cellular and sub-cellular level.

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Lab products found in correlation

Af114, by R&D Systems (2 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Zinc formalin, by Merck Group (1 mentions) Low gelling temperature agarose, by Merck Group (1 mentions) 4 well culture slide, by BD (1 mentions) Stellaris rna fish probe designer, by Biosearch Technologies (1 mentions) Stellaris rna fish probe set, by Biosearch Technologies (1 mentions) Triton x 100, by Merck Group (1 mentions) Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions) Alexa fluor 546 secondary antibody, by Thermo Fisher Scientific (1 mentions) Vectorshield mounting medium, by Vector Laboratories (1 mentions) A 21082, by Thermo Fisher Scientific (1 mentions)

9 protocols using deltavision ultra microscope

Immunostaining of Opa1 Kidney Sections

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The frozen formalin-fixed OCT-embedded sections from control and Opa1^flox/flox:KspCre kidneys samples and the relative controls were dried for 1 h at RT under chemical hood. The OCT was removed through three washings (10 min each) in PBS. The tissue sections were fixed in 4% PFA for 10 min and permeabilized with 0.2% Triton X-100 in PBS. After 1 h blocking in 3% BSA (Sigma-Aldrich, #A7906) 0.1% Triton X-100 in PBS at RT, tissue sections were incubated 1 h at RT or overnight at 4 °C with primary antibody diluted in 3% BSA in PBS. Secondary antibody was incubated with DBA in blocking solution for 1 h at RT. Nuclei were stained with DAPI (1:10,000) in PBS for 10 min at RT. Slides were mounted with Dako Fluorescence Mounting Medium. Representative images were taken using GE Healthcare DeltaVision Ultra microscope.

Steidl M.E., Nigro E.A., Nielsen A.K., Pagliarini R., Cassina L., Lampis M., Podrini C., Chiaravalli M., Mannella V., Distefano G., Yang M., Aslanyan M., Musco G., Roepman R., Frezza C, & Boletta A. (2023). Primary cilia sense glutamine availability and respond via asparagine synthetase. Nature Metabolism, 5(3), 385-397.

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Pluripotency and β-cell Differentiation Imaging

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For pluripotency test, cell lines were cultured in 4-Well Culture Slide (Falcon) until 70% confluence was reached, then fixed with 4% paraformaldehyde. Terminally differentiated β cell aggregates were embedded in 2–4% of low gelling temperature agarose (Sigma-Aldrich), fixed with 10% zinc formalin (Sigma-Aldrich), then included in paraffin. For intracellular staining cells were permeabilized (PermWash 0.4% Triton X-100 in PBS) and stained by using the antibodies listed in the key resources table. All images were acquired using Confocal UltraVIEW ERS microscope (PerkinElmer Life Sciences) and DeltaVision Ultra microscope (GE Healthcare Life Sciences), then analyzed by using Fiji/ImageJ v.1.52p.

Chimienti R., Baccega T., Torchio S., Manenti F., Pellegrini S., Cospito A., Amabile A., Lombardo M.T., Monti P., Sordi V., Lombardo A., Malnati M, & Piemonti L. (2022). Engineering of immune checkpoints B7-H3 and CD155 enhances immune compatibility of MHC-I−/− iPSCs for β cell replacement. Cell Reports, 40(13), 111423.

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CCSER1 mRNA Expression Analysis via Stellaris FISH

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Custom Stellaris FISH Probes were designed based on CCSER1 sequence by using the Stellaris RNA FISH Probe Designer (Biosearch Technologies, Inc.; www.biosearchtech.com). The cells were hybridized with CCSER1 Stellaris RNA FISH Probe set labeled with Q570 dye (Biosearch Technologies, Inc.), following the manufacturer’s instructions (www.biosearchtech.com/stellarisprotocols). Briefly, cells were plated on 13-mm coverslips, and 24 h later, cells were washed twice with PBS, fixed for 10 min with 4% formaldehyde, and washed in PBS. Permeabilization was performed with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min. Cells were then incubated for 5 min in Buffer A (2× SSC, formamide 10%) before the incubation with CCSER1-specific probes (overnight in a wet and dark chamber). Coverslips were washed twice with Buffer A for 30 min at 37°C, twice with PBS, and then nuclei were stained with DAPI for 5 min, washed twice with PBS, and once with Buffer B (2× SSC). Coverslips were finally mounted on microscope slides using Mowiol. Images were acquired with DeltaVision Ultra microscope (GE HealthCare).

Santoliquido B.M., Frenquelli M., Contadini C., Bestetti S., Gaviraghi M., Barbieri E., De Antoni A., Albarello L., Amabile A., Gardini A., Lombardo A., Doglioni C., Provero P., Soddu S., Cittaro D, & Tonon G. (2021). Deletion of a pseudogene within a fragile site triggers the oncogenic expression of the mitotic CCSER1 gene. Life Science Alliance, 4(8), e202101019.

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Immunofluorescence Staining of HEK293 Cells

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HEK293 GLA^−/− cells were transiently transfected as indicated above, grown on coverslip in 24-well plate for 72 h and then fixed in 4% paraformaldehyde (PFA) for 30 min. Cells were permeabilized for 30 s with cold MetOH. After washing in PBS, cells were incubated 30 min at room temperature in 10% preimmune donkey serum-0.1% Triton X-100 in PBS. Cells were then incubated for 1 h at room temperature with the indicated primary antibodies. Cells were then washed in PBS and incubated for 1 h at room temperature with the appropriate secondary antibody: Alexa Fluor 594-conjugated donkey secondary antibody against rabbit immunoglobulin G (IgG) (dilution 1:500; Invitrogen); or Alexa Fluor 488-conjugated donkey secondary antibody against mouse IgG (dilution 1:500; Invitrogen). Cells were then stained for 5 min with 4,6-diamidino-2-phenylindole and mounted using FluorSave Reagent (Calbiochem, San Diego, CA, USA). All slides were acquired by GE healthcare DeltaVision™ Ultra microscope.

Consolato F., De Fusco M., Schaeffer C., Pieruzzi F., Scolari F., Gallieni M., Lanzani C., Feriozzi S, & Rampoldi L. (2022). α-Gal A missense variants associated with Fabry disease can lead to ER stress and induction of the unfolded protein response. Molecular Genetics and Metabolism Reports, 33, 100926.

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Immunohistochemical Analysis of Tumor Vasculature

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Tumors with different treatments were necropsied and incubated overnight on swivel shaker in 50 mM pH7.4 Phosphate buffer with 1% paraformaldehyde, 100 mM Lysine, 2 mg/ml sodium periodate. The samples were washed in PBS for 10 minutes in room temperature for three times. The samples were incubated in PBS with 30% sucrose on swivel shaker for 6–10 hours, then quickly rinsed in PBS and dried completely with a paper towel. Samples were embedded in OCT in tissue cassettes and frozen on dry ice for cutting as 8–10 μm sections on slides. The slides were dried in room temperature for at least 60 minutes, then washed for 5 minutes three times, dried on paper towels and incubated in Rat Immunomix (PBS with 10% Rat serum, 0.05% sodium azide, 0.5% Triton-X-100, and 2 mg/ml BSA for 30 minutes). Then sections were applied in Rat Immunomix overnight with primary antibody anti-CD31 (clone MEC13.3, 1:100). Slides were washed in PBS with 0.1% Tween-20 for 5 minutes three times, washed in PBS for 5 minutes once and fixed in PBS with 1% paraformaldehyde for 2 minutes, washed twice in PBS for 5 minutes and covered by VECTASHIELD Antifade Mounting Medium with DAPI (Vector Lab, Cat. No. H-1200–10). The stained slides were imaged using a DeltaVision™ Ultra microscope from GE Life Sciences and the images were processed by NIH Image J.

Li Y., Su Z., Zhao W., Zhang X., Momin N., Zhang C., Wittrup K.D., Dong Y., Irvine D.J, & Weiss R. (2020). Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate established tumors and prime systemic immunity. Nature cancer, 1(9), 882-893.

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Quantifying Intracellular Localization of GLA

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Z-stacks at 3 μm interval were acquired for each selected field at high magnification (60×) using DeltaVision Ultra microscope (GE healthcare). After acquisition each image was deconvoluted by SoftworX 7.2.0 software (GE healthcare).
Z-stack files were imported into Volocity® software (Quorum technologies, Puslinch, Canada) and for each isoform 20 GLA-expressing cells were analyzed to quantify the entire volume of GLA signal, the entire volume of lysosome marker LAMP1, or the entire volume of ER marker KDEL, and the volume of GLA signal present in lysosomes or ER, expressed as “% of total GLA signal into the lysosome (or ER)”. To calculate the volume of lysosomes, ER or GLA signal, specific regions of interest (ROI) were manually drawn around the cells and a semi-automated threshold was used to quantify positive voxels over background.

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Quantifying Immune Cell Infiltration in Cornea

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Mice were subjected to 5 M NaCl stimulus in the presence of PBS, fosaprepitant 10 mg/mL, or 4 mg/mL oxybuprocaine chloride, as described before in the Acute Corneal Nerve Stimulation Model section. The procedure was repeated 4 times, in 1-hour intervals. One hour after the last stimulus, mice were euthanized and corneas were dissected, washed in PBS, and fixed in acetone at 4°C for 15 minutes. Nonspecific staining was blocked with 2% bovine serum albumin, 5% normal donkey serum followed by immunostaining with goat anti-CD45 (1/200, AF-114; R&D Systems, Minneapolis, MN, USA). After washing with PBS, corneas were incubated with donkey anti-goat Alexa Fluor-546 secondary antibody (1/1000; Invitrogen, Carlsbad, CA, USA) 2 hours at room temperature (RT). Negative control was performed removing the primary antibody. For mounting, Vector Shield mounting medium (Vector Laboratories, Burlingame, CA, USA) was used. Immune cell infiltration was quantified by counting the CD45⁺ cells per field; 6 peripheral and 3 central fields were taken per cornea (20 ×, 5 µm z-stack). Pictures were acquired in a DeltaVision Ultra microscope (GE healthcare, Chicago, IL, USA) and the image analysis was performed using Image J software (National Institutes of Health, Bethesda, MD, USA). Results were expressed as cells/mm².

Lasagni Vitar R.M., Barbariga M., Fonteyne P., Bignami F., Rama P, & Ferrari G. (2021). Modulating Ocular Surface Pain Through Neurokinin-1 Receptor Blockade. Investigative Ophthalmology & Visual Science, 62(3), 26.

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Quantifying Corneal Leukocyte Infiltration

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After 11 days of treatment, corneas were harvested and leukocyte infiltration was quantified. Samples were processed as previously described in section 2.8. Primary immunostaining was performed with goat anti-CD45 (1/200, AF-114; R&D Systems, Minneapolis, MN, USA), followed by incubation with donkey anti-goat Alexa Fluor-546 secondary antibody (1/1000; Invitrogen, Carlsbad, CA, USA) 2 hours at room temperature. Leukocyte infiltration was quantified by counting the CD45+ cells per field. Six peripheral and three central fields were captured per each cornea (20 x, 5 µm z-stack). Images were acquired in a DeltaVision Ultra microscope (GE Healthcare, Chicago, IL, USA). The images were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Results were expressed as cells/fields.

Bonelli F., Campestre F., Lasagni Vitar R.M., Demirsoy I.H., Fonteyne P, & Ferrari G. (2024). Aprepitant Restores Corneal Sensitivity and Reduces Pain in DED. Translational Vision Science & Technology, 13(2), 9.

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Evaluation of NK1R Expression in Epithelial Stem Cells

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To evaluate whether the NK1R was expressed by epithelial stem cell-like cells, we performed a triple staining using p63 and BrdU as previously described earlier. Primary goat anti-NKR1 (1/800, ab61705, Abcam) was co-incubated with p63 antibody for 1 h at RT, followed by Alexa Fluor 633 donkey anti-goat (1/1,000, A21082, Life Technologies) for 1 h at RT. Pictures were acquired in a DeltaVision Ultra microscope (GE healthcare, Chicago, IL, USA) (40×) and image analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). An isotype control was used to establish a fluorescence threshold (Figure S4). Colocalization analysis was performed as described above.

Lasagni Vitar R., Triani F., Barbariga M., Fonteyne P., Rama P, & Ferrari G. (2022). Substance P/neurokinin-1 receptor pathway blockade ameliorates limbal stem cell deficiency by modulating mTOR pathway and preventing cell senescence. Stem Cell Reports, 17(4), 849-863.

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