Prolong diamond antifade mountant

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Japan, Australia

ProLong Diamond Antifade Mountant is a high-performance mounting medium designed for fluorescence microscopy. It is formulated to provide long-lasting protection against photobleaching, preserving fluorescent signals in mounted samples.

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815 protocols using prolong diamond antifade mountant

Multiplexed Immunofluorescence Staining Protocol

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Multiplexed immunofluorescence staining was performed by OPAL 4-colour fluorescence IHC Kit (Perkin Elmer) according to manufacturer’s protocol. The following primary antibodies were used: D240 (M361929-2, DAKO) and IDO (86630, CST). After deparaffinization, steps were repeated for each primary antibody and sections were microwaved in an antigen retrieval buffer for 45 s at 100 °C. The slides were washed and blocked for 10 min at room temperature, and then incubated with a primary antibody. Next, slides were incubated with SuperPicture Polymer Detection Kit-HRP-broad spectrum (Life Technologies), and subsequently treated with Opal fluorochromes (Opal520 and Opal570) diluted 1:150 in an amplification buffer (all provided by the OPAL 4-colour fluorescence IHC kit) for 10 min at room temperature. Finally, a microwave treatment with AR6 buffer was performed. The slides were then incubated with a 4′,6-diamidino-2-phenylindole (DAPI) working solution (provided by the OPAL 4-colour fluorescence IHC kit) for 5 min at room temperature and then mounted under coverslips with ProLong Diamond Antifade Mountant (Life Technologies).

Zhou C., Zhang Y., Yan R., Huang L., Mellor A.L., Yang Y., Chen X., Wei W., Wu X., Yu L., Liang L., Zhang D., Wu S, & Wang W. (2020). Exosome-derived miR-142-5p remodels lymphatic vessels and induces IDO to promote immune privilege in the tumour microenvironment. Cell Death and Differentiation, 28(2), 715-729.

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RNAscope for Brain Tissue Analysis

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RNAscope (Advanced Cell Diagnostics, ACD) was performed as follows. Briefly, brains were quickly frozen in Optimum Cutting Temperature compound (Tissue-Tek), using isopentane chilled with liquid nitrogen. Ten-micrometer thick brain slices were prepared using a NX70 cryostat (Thermo Fisher Scientific). Sections were subsequently fixed in ice-cold 4% paraformaldehyde for 30 min. Sections were then dehydrated using a series of ethanol solutions (50–100%), before drying and incubating with Protease IV for 20 min at room temperature. Slides were washed in phosphate-buffered saline and hybridized with gene-specific probes (Supplementary Table 5) for 2 h at 40 °C in a HybEZ Oven (ACD). Non-annealed probes were removed by washing sections in 1× proprietary wash buffer. Probes were then detected via sequential hybridization of proprietary amplifiers and labeled “secondary” probes (Amp 1–Amp 4). Finally, sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted using ProLong Diamond Antifade Mountant (Life Technologies).

Batiuk M.Y., Martirosyan A., Wahis J., de Vin F., Marneffe C., Kusserow C., Koeppen J., Viana J.F., Oliveira J.F., Voet T., Ponting C.P., Belgard T.G, & Holt M.G. (2020). Identification of region-specific astrocyte subtypes at single cell resolution. Nature Communications, 11, 1220.

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Analysis of Autophagy Protein Interactions

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Cells were plated on glass cover slides in complete medium and incubated overnight at 37 °C and 5% CO₂. Cells were then treated with starvation medium for autophagy stimulation. Cells were then fixed and permeabilized as described above. After a PBS wash, cells were incubated with appropriate combination of rabbit anti-LC3 and goat anti-MLP primary antibodies diluted in PBS-1% BSA at 4 °C. PLAs were performed using a Duolink (Sigma-Aldrich) kit according to the manufacturer’s instructions.
Nuclei were stained with Sytox green (Life Technologies) 50 nm for 10 min. Coverslips were mounted on antifade mountant (ProLong Diamond Antifade Mountant, Life Technologies). Fluorescence intensity was visualized with an LSM 510 confocal microscope (Zeiss). Images were analyzed in ImageJ to calculate the density of PLA puncta.

Rashid M.M., Runci A., Polletta L., Carnevale I., Morgante E., Foglio E., Arcangeli T., Sansone L., Russo M.A, & Tafani M. (2015). Muscle LIM protein/CSRP3: a mechanosensor with a role in autophagy. Cell Death Discovery, 1, 15014-.

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Immunostaining Protocol for Cellular Imaging

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For immunostaining, cells were grown on coverslips or directly on culture slides (for Laser microirradiation). Cells were pre-extracted with 0.25–0.125% Triton X-100 in PBS on ice, and then fixed with 3% paraformaldehyde in 2% sucrose solution for 15 minutes at room temperature. Then cells were permeabilized with ice cold 0.5% Triton X-100 in PBS on ice for 5 minutes, blocked in 3% BSA and 0.05% Tween-20 in PBS prior to incubation with primary antibodies. After 3 washes with 0.05% Tween-20 in PBS, cells were incubated with secondary antibodies. For nuclei staining, 1 µg/mL DAPI solution was used. Coverslips mounted onto microscope slides using ProLong® Diamond Antifade Mountant (Life Technologies). All the antibodies used are listed in Table S1.

Lashgari A., Fauteux M., Maréchal A, & Gaudreau L. (2018). Cellular Depletion of BRD8 Causes p53-Dependent Apoptosis and Induces a DNA Damage Response in Non-Stressed Cells. Scientific Reports, 8, 14089.

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Immunofluorescence Protocol for Cell Fixation

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Cells were fixed with 4% buffered paraformaldehyde for 15 min at room temperature, permeabilised with 0.5% Triton X-100 in PBS for 10 min and blocked with 3% BSA and 0.1% Tween 20 in PBS for 30 min at room temperature. Incubation with primary antibodies (Table S3C) diluted in PBS with 0.1% Triton X-100 and 3% donkey serum was overnight at 4°C and secondary antibodies were added for 1 h at room temperature. Slides were mounted with Prolong Diamond Antifade Mountant (Life Technologies).

Guo G., von Meyenn F., Rostovskaya M., Clarke J., Dietmann S., Baker D., Sahakyan A., Myers S., Bertone P., Reik W., Plath K, & Smith A. (2017). Epigenetic resetting of human pluripotency. Development (Cambridge, England), 144(15), 2748-2763.

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3D Culturing of Melanoma Spheroids in NFC Hydrogel

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Sterile GrowDex™ nanofibrillar cellulose (NFC) hydrogel was obtained from UPM-Kymmene Corporation, Finland. The NFC concentration of the hydrogel was 1.55 wt%. To determine the optimal conditions for 3D cell culturing, three concentrations (0.4%; 0.7% and 1% wt/v) of NFC hydrogel, as well as two cell-seeding concentrations (50,000 and 100,000 cells per 100 ul of NFC / 96-well) were tested. MUG-Mel2 spheroids grown in 0.4% NFC hydrogel were collected at day three for live calcein staining. Prior to collection, NFC hydrogel was digested from the cell culture by cellulase enzyme (VTT, Finland) for 24 h at 37 °C using 300 µg of cellulose per 1 mg of NFC. Spheroids were washed three times with 1xDPBS, suspended in staining solution containing Calcein-AM 1:500 (Cellstain double staining kit; Sigma Aldrich) and 50 µg/ml DAPI (Invitrogen, Carlsbad, CA) for 15 min at 37 °C, and subsequently mounted using Prolong Diamond Antifade Mountant (Life Technologies). Fluorescent images were taken with the Leica TCS SP5 microscope using HCX PL APO 20x/0.7 objective. Imaris 8.4.1 software was used for image acquisition.

Rinner B., Gandolfi G., Meditz K., Frisch M.T., Wagner K., Ciarrocchi A., Torricelli F., Koivuniemi R., Niklander J., Liegl-Atzwanger B., Lohberger B., Heitzer E., Ghaffari-Tabrizi-Wizsy N., Zweytick D, & Zalaudek I. (2017). MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line. Scientific Reports, 7, 2098.

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Automated Multiplex Immunofluorescence Imaging

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Automated mIF was performed as previously described (Giraldo et al. 2018 (link); Davis et al. 2020 (link)). Briefly, slides were heated and dewaxed to remove any paraffin. Antigen retrieval was performed using ER2 followed by washing steps. Nonspecific staining was blocked using Blocking/Ab Diluent (Akoya Biosciences) followed by the first primary antibody (see position 1 in Supplemental Table S3). The corresponding polymer was applied followed by the tyramide signal amplification dye (Opal Automation Multiplex IHC Kit; Akoya Biosciences). Slides were heated to strip the primary antibody and polymer, washed, and blocked again. The process was repeated for positions 2–6. After the last step of antibody striping, the slides were stained for DAPI and coverslipped using ProLong Diamond Antifade Mountant (Life Technologies).
Slides were scanned using the Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences). A 10× (1 µm/px) whole-slide scan was acquired and used as a guide to select 20 high power field (HPF) for 20× image acquisition. These 20× HPF images were processed in Inform software (Akoya Biosciences) and exported to images with QPTIFF format.

Sena L.A., Salles D.C., Engle E.L., Zhu Q., Tukachinsky H., Lotan T.L, & Antonarakis E.S. (2021). Mismatch repair–deficient prostate cancer with parenchymal brain metastases treated with immune checkpoint blockade. Cold Spring Harbor Molecular Case Studies, 7(4), a006094.

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Immunostaining of Embryonic Tissues

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Embryos were collected at E8.0-E8.5 and fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature. Embryos were washed 3 times (15 min each) in phosphate buffered saline (PBS), then blocked with 1% goat serum and 0.1% Triton-X in PBS (PBGT) for 1 h at room temperature. Primary antibodies in PBGT were added and incubated overnight at 4 °C. After 315 min washes in PBGT, secondary antibodies in PBGT were added for 2 h at room temperature. Then, samples were washed 3 times (15 min each) in PBS and 0.1% Triton-X, and mounted on slides (Denville) using ProLong Diamond Antifade Mountant (Life Technologies). Antibodies used are available in Supplementary Material, Table S2.
Alternatively, embryos previously dehydrated in methanol series were rehydrated, blocked in PBGT, and the protocol was followed as described above.
To acquire sections through the midline, embryos were stained as wholemount and imaged, then rinsed 3 times in PBS and 0.1% Triton-X, dehydrated in 30% sucrose, embedded in OCT and cryosectioned. Coverslips were applied to sections with ProLong Diamond Antifade Mountant.

Shylo N.A., Emmanouil E., Ramrattan D, & Weatherbee S.D. (2019). Loss of ciliary transition zone protein TMEM107 leads to heterotaxy in mice. Developmental biology, 460(2), 187-199.

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Visualization of Lymph Node Stromal Cells

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For analysis of LN stromal cells by confocal microscopy, LNs were isolated on day 7 after transplantation and frozen in OCT (Cellpath). Sections (8 μm) were cut on a cryostat, dried, and fixed with acetone (–20°C). Primary antibodies were CD31-FITC (clone MEC13.3, BD Biosciences) and gp38-biotin (eBio8.1.1, eBioscience). Secondary antibodies were anti-FITC Alexa 488 (Life Technologies) and Streptavidin eFluor 570 (eBioscience). Sections were stained with DAPI and mounted with ProLong Diamond Antifade Mountant (Life Technologies). All images were captured on a Nikon Ti inverted microscope using a C2 confocal scan head (Nikon Instruments). Images were acquired with a 40× (Plan Apochromat N.A. 0.095 W.D. 0.21 mm) objective. Image analysis was done using the software ImageJ (NIH).

Dertschnig S., Evans P., Santos e Sousa P., Manzo T., Ferrer I.R., Stauss H.J., Bennett C.L, & Chakraverty R. (2020). Graft-versus-host disease reduces lymph node display of tissue-restricted self-antigens and promotes autoimmunity. The Journal of Clinical Investigation, 130(4), 1896-1911.

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Immunofluorescence Staining Protocol

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Cell fixation and antibody staining were performed as previously described^{7 (link)}. Briefly, cells were fixed with ice-cold (−30 C) methanol for 15 minutes – when staining for centromeres, centrosomes, cGAS, Vimentin, β-actin, IRF3, or a-tubulin – or 4% paraformaldehyde – when staining for RelB, p65, STING, ssDNA, dsDNA, CoxIV, or β-catenin. Subsequently, cells were permeabilized using 1% triton for 4 minutes. See supplementary Table 1 for antibody information. For selective plasma membrane permeabilization used for cytosolic dsDNA and ssDNA staining, cells were treated with 0.02% saponin for 5 minutes after fixation. For single-stranded (Thermo Fisher FEREN0321) and double stranded (Life Technologies – EN0771)-specific nuclease treatment, cells were also permeabilized with 0.02% saponin for 2 minutes and treated with either nucleases for 10 minutes before fixation using 4% paraformaldehyde. TBS-BSA was used as a blocking agent during antibody staining. DAPI was added together with secondary antibodies. Cells were mounted with Prolong Diamond Antifade Mountant (Life Technologies – P36961). cGAMP (Invivogen – tlrl-nacga23) was transfected into cells at a concentration of 4μg/ml using lipofectamine2000 that was added for 3–4 hours and then replaced with regular serum containing medium.

Bakhoum S.F., Ngo B., Laughney A.M., Cavallo J.A., Murphy C.J., Ly P., Shah P., Sriram R.K., Watkins T.B., Taunk N.K., Duran M., Pauli C., Shaw C., Chadalavada K., Rajasekhar V.K., Genovese G., Venkatesan S., Birkbak N.J., McGranahan N., Lundquist M., LaPlant Q., Healey J.H., Elemento O., Chung C.H., Lee N.Y., Imielenski M., Nanjangud G., Pe’er D., Cleveland D.W., Powell S.N., Lammerding J., Swanton C, & Cantley L.C. (2018). Chromosomal instability drives metastasis through a cytosolic DNA response. Nature, 553(7689), 467-472.

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