Diamond antifade mounting medium

Manufactured by Thermo Fisher Scientific

Sourced in Japan

Diamond antifade mounting medium is a reagent used in microscopy applications to preserve and protect fluorescent samples mounted on microscope slides. It helps prevent the fading or bleaching of fluorescent dyes and proteins, allowing for improved imaging and analysis of the sample.

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Lab products found in correlation

Spectral dapi, by Akoya Biosciences (4 mentions) Bond rxm research stainer, by Leica (4 mentions) Opal polymer hrp secondary antibody, by Akoya Biosciences (3 mentions) Opal fluorophore, by Akoya Biosciences (3 mentions) Ae1 ae3, by Agilent Technologies (2 mentions) Ab108986, by Abcam (1 mentions) Plan apo 60, by Nikon (1 mentions) A1 confocal laser microscope system, by Nikon (1 mentions) Plan apo 20, by Nikon (1 mentions) Model eclipse ni u, by Nikon (1 mentions) Dylight 594, by Vector Laboratories (1 mentions) Chicken anti gfap, by Novus Biologicals (1 mentions) Rat anti cd45, by Abcam (1 mentions) Goat anti iba 1, by Novus Biologicals (1 mentions) Guinea pig anti neun, by Synaptic Systems (1 mentions) Rabbit anti pgp9, by Abcam (1 mentions) Cytm3 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions) Cytm3 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions) Vectra polaris automated quantitative pathology imaging system, by Akoya Biosciences (1 mentions) D5d5r, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Mounting medium (119 citations) Prolong Diamond Antifade mounting medium (73 citations) Antifade mounting medium (65 citations) ProLong diamond Antifade Mounting medium with DAPI (16 citations) AntiFade medium (12 citations) Slow Fade Diamond Antifade mounting medium (4 citations)

9 protocols using diamond antifade mounting medium

Immunohistochemical Analysis of Prostate Samples

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At the endpoint of CP1 instillation experiment, the mice were euthanized, and the prostates were harvested from mice as described previously (34 (link)). Prostate samples were fixed in 10% formalin, processed, and embedded in paraffin by the Northwestern University Mouse Histology and Phenotyping Core facility. The formalin-fixed paraffin-embedded (FFPE) samples were then sectioned (5 μm sections) and mounted on glass slides for staining.
The antibodies used in this study were as follows: rabbit anti-PGP9.5 (1:500, catalog # ab108986, RRID: AB_10891773; abcam, Cambridge, UK) and mouse anti-human mast cell tryptase (1:500, with cross-reactivity with mouse, catalog # 369402, RRID: AB_2566541; BioLegend®, San Diego, CA). The immunolabeling was visualized by using Cy^tm2-conjugated donkey anti-mouse IgG and Cy^tm5-conjugated-donkey anti-rabbit IgG and mounted using diamond Antifade mounting medium (Invitrogen, Thermo Fisher, Hampton, NH).
Images were obtained on a Nikon A1 Confocal Laser Microscope System (Plan Apo 20 × NA 0.75 and Plan Apo 60 × NA1.4 Oil, objectives). Images were taken on the same confocal imaging settings as the template for acquiring all images.

Pattabiraman G., Liu Z., Paul M., Schaeffer A.J, & Thumbikat P. (2022). mMCP7, a Mouse Ortholog of δ Tryptase, Mediates Pelvic Tactile Allodynia in a Model of Chronic Pelvic Pain. Frontiers in Pain Research, 2, 805136.

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Quantifying Duodenal PMCA1 Expression

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Duodenal segments were fixed overnight in 0.1 M phosphate-buffered saline (PBS) containing 4% paraformaldehyde. After being embedded in paraffin, the specimens were cut longitudinally into 4-µm sections which were later incubated at 95 °C for 20 min in sodium citrate buffer solution (pH 6.0). Non-specific bindings were blocked for 2 h by 4% bovine serum albumin, 5% normal goat serum, and 0.1% Tween-20 in PBS. Thereafter, sections were incubated at 4 °C overnight in moist chamber with rabbit monoclonal anti-PMCA1 (plasma membrane Ca²⁺ ATPase) at 1:500 dilution (Cat# ab190355, Abcam, RRID:AB_2893200). After being washed with 0.5% Tween-20 in PBS, sections were incubated for 1 h at room temperature in the dark with anti-rabbit Dylight 594 (Cat#DI-1594, Vector Laboratories) in blocking buffer. Sections were mounted with slow fade Diamond antifade mounting medium containing DAPI (Cat# S36964, Invitrogen) visualized under a fluorescent microscope (model eclipse Ni-U; Nikon). Fluorescent intensity was measured from 5 images per rat, 3–4 villi per image (total of 15–20 villi/rat), 5 rats/group by NIS-elements BR imaging software (version 4.0).

Suntornsaratoon P., Thongklam T., Saetae T., Kodmit B., Lapmanee S., Malaivijitnond S., Charoenphandhu N, & Krishnamra N. (2023). Running exercise with and without calcium supplementation from tuna bone reduced bone impairment caused by low calcium intake in young adult rats. Scientific Reports, 13, 9568.

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Immunohistochemical Analysis of Chronic Pain

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On 14 and 28 days after induction of EAP based on pelvic tactile allodynia response, mice were anesthetized with 2.5% isoflurane (Isothesia; Butler) and transcardially perfused with cold PBS followed by ice-cold 4% paraformaldehyde. DRGs, spinal cords of cervical, lumbar and sacral segments and prostates were removed and post-fixed overnight in 4% paraformaldehyde. Then the tissues were transferred to 30% sucrose solution overnight. Tissues were mounted with ornithine carbamyl transferase embedding medium (Tissue Tek) on dry ice and stored at −80°C for further experiments. The antibodies used in this study were as follows: rabbit anti-TMEM199 (1:1,000, Abcam), goat anti-IBA1 (1:1,000, Novus), rat anti-CD45 (1:500, Abcam), guinea pig anti-NeuN (1:1,000, Synaptic Systems), rabbit anti-PGP9.5 (1:500, Abcam), mouse anti-CCL2 (1:200, Proteintech) and chicken anti-GFAP (1:1,000, Novus). The multiple immunolabelings were visualized respectively by using Cy^tm2- or Cy^tm3-conjugated-donkey anti-rabbit IgG, Cy^tm3-conjugated-donkey anti-mouse IgG, Cy^tm3 or 5-conjugated-donkey anti-goat IgG, Cy^tm3-conjugated-donkey anti-rat IgG, Cy^tm5-conjugated-donkey anti-Guinea Pig IgG, Brilliant Violet 421-conjugated Donkey Anti-Chicken IgG (1:1,000, Jackson ImmunoResearch). Diamond Antifade mounting medium (Invitrogen) with or without DAPI (dependent on wavelength assignment in multicolor labeling).

Liu Z., Murphy S.F., Wong L., Schaeffer A.J, & Thumbikat P. (2020). Neuronal/astrocytic expression of chemokine (C-C motif) Ligand 2 is associated with monocyte/macrophage recruitment in male chronic pelvic pain. Pain, 161(11), 2581-2591.

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Multiplexed Immunofluorescence Staining of FFPE Tumor Samples

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Tumor samples were fixed in formalin and embedded in paraffin (FFPE) and 4 μm thick sections were cut for staining. A pathologist (AW) evaluated hematoxylin and eosin (H&E) stained slides from each FFPE block. MIF staining was performed on the BOND RX^m Research Stainer (Leica Biosystems). The MIF panel consisted of the following antibodies (clone, company, and opal fluorophores): TIM3 (D5D5R™, Cell Signaling, Opal 540), CTLA4 (UMAB249, Biocare Medical, Opal 570), OX40 (E9U7O, Cell Signaling, Opal 520), PD-L1 (E1L3N, Cell Signaling, Opal 620), LAG3 (D2G4O, Cell Signaling, Opal 650), pancytokeratin (AE1AE3, Agilent DAKO, Opal 690). All AKOYA reagents used for MIF staining are part of a detection kit (NEL821001KT). Spectral DAPI (AKOYA Biosciences) was applied once slides were removed from the BOND. They were cover slipped using an aqueous method and Diamond antifade mounting medium (Invitrogen, ThermoFisher). Slides were imaged on the Vectra^® Polaris Automated Quantitative Pathology Imaging System (AKOYA Biosciences). Spectral unmixing, tissue and cell segmentation, and cell phenotyping were performed on inForm^® Software v2.4.8 (AKOYA Biosciences).

Hellström I., Ledbetter J.A., Scholler N., Yang Y., Ye Z., Goodman G., Pullman J., Hayden-Ledbetter M, & Hellström K.E. (2001). CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer. Proceedings of the National Academy of Sciences of the United States of America, 98(12), 6783-6788.

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Multiplex Immunofluorescence for Cell Cycle Markers

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Formalin-fixed Paraffin-embedded (FFPE) tissue sections orTMAs were cut at 4–5 µm on charged slides. Slides were dried at 65°C for 6 hours. After drying, the slides were placed on the BOND RXm Research Stainer (Leica Biosystems) and deparaffinized with BOND Dewax solution (AR9222, Lecia Biosystems). The multispectral immunofluorescent (mIF) staining process involved serial applications of the following for each biomarker: epitope retrieval/stripping with ER1 (citrate buffer pH 6, AR996, Leica Biosystems ), blocking buffer (AKOYA Biosciences), primary antibody, Opal Polymer HRP secondary antibody (AKOYA Biosciences), Opal Fluorophore (AKOYA Biosciences). All AKOYA reagents used for mIF staining come as a kit (NEL821001KT). Spectral DAPI (AKOYA Biosciences) was applied once slides were removed from the BOND. They were cover slipped using an aqueous method and Diamond antifade mounting medium (Invitrogen ThermoFisher). The mIF panel consisted of the following antibodies (clone, company, and Opal Fluorophores): Cyclin D (SP4, Epredia, Opal 570), Cyclin E (EP435E, abcam, Opal 520), MCM2 (RBT-MCM2, Biosb, Opal Polaris 480), Pan Cytokeratin (AE1AE3, Agilent DAKO, Opal Polaris 780), pHH3 (Ser10, Millipore Sigma, Opal 620), pRB (Ser807/811, Cell Signaling, Opal 690).

Knudsen E.S., Kumarasamy V., Nambiar R., Pearson J.D., Vail P., Rosenheck H., Wang J., Eng K., Bremner R., Schramek D., Rubin S.M., Welm A.L, & Witkiewicz A.K. (2022). CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities. Cell reports, 38(9), 110448.

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Multiplex Immunofluorescence Staining of FFPE Sections

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Formalin-fixed paraffin-embedded (FFPE) 4 µm sections were cut and placed on charged slides. Slides were dried at 65 °C for 2 h. After drying, the slides were placed on the BOND RX^m Research Stainer (Leica Biosystems) and deparaffinized with BOND Dewax solution (AR9222, Lecia Biosystems). The multispectral immunofluorescent (mIF) staining process involved serial repetitions of the following for each biomarker: epitope retrieval/stripping with ER1 (citrate buffer pH 6, AR996, Leica Biosystems) or ER2 (Tris-EDTA buffer pH9, AR9640, Leica Biosystems), blocking buffer (AKOYA Biosciences), primary antibody, Opal Polymer HRP secondary antibody (AKOYA Biosciences), Opal Fluorophore (AKOYA Biosciences). All AKOYA reagents used for mIF staining come as a kit (NEL821001KT). Spectral DAPI (AKOYA Biosciences) was applied once slides were removed from the BOND. They were cover slipped using an aqueous method and Diamond antifade mounting medium (Invitrogen ThermoFisher). The mIF panel consisted of the following antibodies: Ki67 (Abcam, ab16667), AE1AE3 (Dako, M3515), CCNE (Abcam, ab33911), CCND1 (ThermoFisher, MA1-39546), CCNA (Abcam, ab32386), RB (Cell Signaling, 9309s), pRB (Cell Signaling, 8516), and MCM2 (BioSb, BSB6334).

Witkiewicz A.K., Schultz E., Wang J., Hamilton D., Levine E., O’Connor T, & Knudsen E.S. (2023). Determinants of response to CDK4/6 inhibitors in the real-world setting. NPJ Precision Oncology, 7, 90.

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Multiplex Immunofluorescence for Cell Cycle Markers

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Assessing Viability of Adherent Cells

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The LIVE/DEAD™ Viability/Cytotoxicity Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used per the manufacturer’s directions. At 24 h post isolation, unfixed ACMs were labeled with 2 μM calcein-AM (green) and 2 μM ethidium homodimer-1 (red) for 40 min. Cells were then fixed with 10% formalin solution and mounted with Diamond Antifade Mounting Medium (Thermo Fisher Scientific). Images were acquired using an Olympus BX51 fluorescence microscope and quantified with ImageJ. Ten random fields of view were analyzed per substrate condition. Analysis was performed by taking the ratio of ethidium homodimer-1-positive cells over the total number of platted ACMs/field.

Zhai R., Varner E.L., Rao A., Karhadkar S., Di Carlo A., Snyder N.W, & Sato P.Y. (2022). Myocardial GRK2 Reduces Fatty Acid Metabolism and β-Adrenergic Receptor-Mediated Mitochondrial Responses. International Journal of Molecular Sciences, 23(5), 2777.

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Quantification of NEAT1 RNA Foci in iPS-CMs

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A set of Quasar670-conjugated RNA FISH probes were designed for NEAT1 using the Stellaris Probe Designer v. 4.2 and acquired from LGC Biosearch (Petaluma, CA, USA). RNA FISH was performed according to the manufacturer’s protocol. iPS-CM were seeded and cultured on glass coverslips in a 12-well plate. Five days after seeding, cells were fixed, permeabilized, and incubated with hybridization buffer containing 125 nM FISH probes overnight. The following day, cells were counterstained with 150 nM 4′,6-diamidino-2-phenylindole (DAPI) for 30 min, washed and mounted on microscopy slides with Diamond antifade mounting medium (Thermo Fisher) and visualized with a Nikon LU4A Ti Microscope (Nikon Instruments, Tokyo, Japan). The number of distinct NEAT1 foci/cell was enumerated in >20 individual images from at least two different slides per condition, blinded to experimental groups.

Gidlöf O., Bader K., Celik S., Grossi M., Nakagawa S., Hirose T., Metzler B., Olde B, & Erlinge D. (2020). Inhibition of the long non-coding RNA NEAT1 protects cardiomyocytes from hypoxia in vitro via decreased pri-miRNA processing. Cell Death & Disease, 11(8), 677.

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