Prolong glass antifade mounting medium

Manufactured by Thermo Fisher Scientific

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ProLong glass antifade mounting medium is a product designed for use in fluorescence microscopy. It is formulated to reduce photobleaching of fluorescent dyes, enabling prolonged observation of labeled samples.

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12 protocols using prolong glass antifade mounting medium

Cellular Localization of TAC Chimeras

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To determine the subcellular localization of the TAC chimeras or spike glycoprotein, the various constructs were transfected into HeLa cells on sterile glass coverslips using jetOPTIMUS transfection reagent according to the manufacturer’s protocol. Cells were fixed the following day with 4% formaldehyde (Sigma-Aldrich) for 10min, permeabilized and blocked with PBS containing 0.4% (v/v) Triton X-100% and 2% immunoglobulinG-free BSA (Jackson ImmunoResearch) for 1 hr, and then probed with the indicated antibodies in PBS containing 0.1% Triton X-100% and 0.5% BSA. Following fluorophore-conjugated secondary antibody treatment and washing, the processed cells were mounted in ProLong® Glass antifade mounting medium (Life Technologies), and the images were acquired with an LSM880 confocal microscope (Carl Zeiss Inc., Peabody, MA). Images were analyzed by ImageJ software (Fiji).

Jennings B.C., Kornfeld S, & Doray B. (2021). A weak COPI binding motif in the cytoplasmic tail of SARS‐CoV‐2 spike glycoprotein is necessary for its cleavage, glycosylation, and localization. Febs Letters, 595(13), 1758-1767.

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Subcellular Localization of PTase Constructs

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To determine the subcellular localization of either WT or mutant PTase, the various constructs were transfected into HeLa cells on sterile glass coverslips using jetOPTIMUS transfection reagent. Cells were fixed the following day with 4% formaldehyde (Sigma-Aldrich) for 10 min, then permeabilized and blocked with PBS containing 0.4% (v/v) Triton X-100% and 2% immunoglobulin G-free BSA (Jackson ImmunoResearch) for 1 hr. Cells were probed with the indicated antibodies in PBS containing 0.1% Triton X-100% and 0.5% BSA. For primary antibodies, the anti-V5 mouse monoclonal antibody (Invitrogen) was used at a dilution of 1:1000 while the anti-giantin rabbit polyclonal antibody (BioLegends) was used at a dilution of 1:2000. For secondary antibodies, the goat anti-mouse Alexa 488+ antibody (Invitrogen) and the goat anti-rabbit Alexa 555 antibody (Invitrogen) were both used at a dilution of 1:750. Following fluorophore-conjugated secondary antibody treatment and washing, the processed cells were mounted in ProLong® Glass antifade mounting medium (Life Technologies), and the images were acquired with an LSM880 confocal microscope (Carl Zeiss Inc., Peabody, MA). Images were analyzed by ImageJ software (Fiji, version 1.53n).

Li H., Lee W.S., Feng X., Bai L., Jennings B.C., Liu L., Doray B., Canfield W.M., Kornfeld S, & Li H. (2022). Structure of the human GlcNAc-1-phosphotransferase αβ subunits reveals regulatory mechanism for lysosomal enzyme glycan phosphorylation. Nature structural & molecular biology, 29(4), 348-356.

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Quantifying Filopodia Dynamics in MDA-MB231 Cells

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MDA-MB231 cells were cultured in 24-well plates containing glass coverslips. When 60% confluent, cells were serum starved for 16 h, preincubated with 50 μM compound NCGC00131308 for 2 h, then incubated with 100 ng/mL epidermal growth factor (EGF) or vehicle for 10 min. Cells were fixed with 4% PFA and stained with Alexa Fluor 488 Phalloidin (Invitrogen™#A12379) and Hoechst 33342 (BD Biosciences #561908) to visualize F-actin and DNA, respectively. Coverslips were mounted with ProLong glass antifade mounting medium (Invitrogen), then imaged with a Zeiss LSM880 confocal microscope using a 63× objective lens. Cells and filopodia were segmented using FIJI/ImageJ^{49 (link)}. Individual filopodia were segmented using the FiloQuant FIJI plugin^{50 (link)}. Cells were segmented by Voronoi tessellation using Hoechst 33342 stained nuclei as seed points. The segmented images were quantified using the scikit-image Python package^{51 (link)}. The following geometric and spatial parameters of the filopodia were quantified: (1) the branch length of each filopodial segment, (2) cumulative area of each segment, (3) eccentricity, defined as the ratio of the minor axis length to the major axis length, and (4) the number of filopodial segments per unit exposed cell membrane. Further data handling and plotting were done using the tidyverse R package. At least 200 cells were analyzed for each condition.

Sayedyahossein S., Smith J., Barnaeva E., Li Z., Choe J., Ronzetti M., Dextras C., Hu X., Marugan J., Southall N., Baljinnyam B., Thines L., Tran A.D., Ferrer M, & Sacks D.B. (2022). Discovery of small molecule inhibitors that effectively disrupt IQGAP1-Cdc42 interaction in breast cancer cells. Scientific Reports, 12, 17372.

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Immunofluorescence Imaging of Mitochondrial Proteins

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U2OS cells were grown on 12 mm round glass coverslips (#1.5) and stained for 30 min with 100 nM of Mitotracker DeepRed (Invitrogen, Carlsbad, CA, cat# M22426), washed with PBS, and fixed in 4% PFA in PBS for 20 min at room temperature. Cells were washed again with PBS, permeabilized for 10 min with 0.1% Triton X-100 in PBS, blocked with 5% bovine serum albumine (BSA) in PBS for 1 hr at room temperature, and immunolabeled with rabbit anti-OCIAD1 (Invitrogen, cat# PA5-20834, 1:10000) or rabbit anti-OCIAD2 (Invitrogen, cat# PA5-59375, 1:5000) antibodies for 1 hr at room temperature in 1% BSA in PBS. Cells were washed again in PBS and incubated with donkey anti-rabbit IgG conjugated with AlexaFluor 488 (Invitrogen, Carlsbad, CA, cat# A21206, 1:1000) in 1% BSA in PBS for 1 hr at room temperature. Finally, cells were washed again in PBS and mounted on glass slides with ProLong Glass antifade mounting medium (Invitrogen, Carlsbad, CA, cat# P36980). Images were collected using the spinning disk module of a Marianas SDC Real Time 3D Confocoal-TIRF microscope (Intelligent Imaging Innovations; Denver, CO) fitted with a 100×, 1.46 NA objective and a Hamamatsu (Japan) Orca Flash 4.0 sCMOS camera. Images were captured with SlideBook (Intelligent Imaging Innovations) and linear adjustments were made using ImageJ.

Le Vasseur M., Friedman J., Jost M., Xu J., Yamada J., Kampmann M., Horlbeck M.A., Salemi M.R., Phinney B.S., Weissman J.S, & Nunnari J. (2021). Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells. eLife, 10, e67624.

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Immunofluorescence Analysis of Irradiated NPC Cells

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For immunofluorescence, NPC cells transfected with plasmids were seeded on a confocal dish (Cat no.: 801002, NEST, CHN), exposing to 8 Gy of irradiation when cells were grown to ~70% confluency. The cells were harvested at 0, 1, 2, and 4 h and then fixed, permeabilized, and blocked by using the Image-iT^TM Fixation/ Permeabilization Kit (Invitrogen, Carlsbad, CA, USA). Blocked cells were incubated overnight with the appropriate primary antibody at 4 °C. After washing in cold PBS three times, Invitrogen Alexa Fluor Plus 594 goat anti-rabbit IgG secondary antibody or Alexa Fluor Plus 488 goat anti-mouse IgG secondary antibody were incubated. Subsequently, cells were washed and sealed with an Invitrogen ProLong™ glass antifade mounting medium. NucBlue™ Stain was used to counterstain nuclei. All immunofluorescence images were obtained and visualized by LSM 900 (Zeiss, Jena, Germany) confocal microscope using a 63×oil immersion objective lens. Results were analyzed by Image J (Media Cybernetics, Inc., Rockville, MD, USA).

Wang Y.H., Guo Z., An L., Zhou Y., Xu H., Xiong J., Liu Z.Q., Chen X.P., Zhou H.H., Li X., Liu T., Huang W.H, & Zhang W. (2021). LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer. Cell Death & Disease, 12(5), 454.

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Microscopic Imaging of GBS Infection

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Fifty thousand monocytes were infected as described above with GFPmut3-GBS^{24 (link)} or mCherry-GBS^{22 (link)} for 1 h, followed by application of antibiotics as described above. Then, 5 h later, the infected cells were subjected to three washes of PBS and fixed for 15 min at 37 °C using 3.5% (wt/vol) paraformaldehyde. The cells were concentrated by centrifugation and mounted using ProLong™ Glass antifade mounting medium (Invitrogen). The cells were visualized using a Zeiss AxioImager.M2 microscope (Carl Zeiss MicroImaging) fitted with a Plan-Apochromat X63/1.40 lens objective and AxioCam MRm Rev.3 and MRc 5 cameras. Images of cells were captured with 63HE and 44 filter sets (to detect mCherry [587 nm, 610 nm], GFPmut3 [494 nm, 518 nm] fluorescence, respectively, with excitation and emission spectra listed consecutively for each) and phase contrast with Zen Pro (version 2) software. Composite images were captured as a collection of 20–26 Z-stacked micrographs covering a total depth of approximately 8–10 μm, which were compiled using maximum intensity projection to generate two-dimensional images.

Sullivan M.J., Prince D., Goh K.G., Katupitiya L., Gosling D., Crowley M.R., Crossman D.K, & Ulett G.C. (2023). Dual RNA sequencing of group B Streptococcus-infected human monocytes reveals new insights into host–pathogen interactions and bacterial evasion of phagocytosis. Scientific Reports, 13, 2137.

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Super-Resolution Imaging of Synaptic Proteins

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Tissue sections prepared for super resolution imaging were mounted on charged slides as described and coverslipped with ProLong Glass Antifade mounting medium (P36980, Invitrogen) using 170 ± 5 µm No. 1.5H High Precision cover glasses. Bassoon- and PSD95-immunopositive puncta were captured using the Zeiss Elyra 7 super resolution microscope with Lattice SIM2. Images were acquired using a 63 × oil objective. 3.91 µm z-stacks were imaged at a step size of 0.126 µm, with laser power 0.9% and 90 ms exposure, and subsequently processed using the SIM2 ‘Low contrast’ reconstruction settings. The total numbers of Bassoon- and PSD95-puncta per z-stack were determined by thresholding using Imaris Software and normalized per mm³ of tissue.

Mate De Gerando A., Welikovitch L.A., Khasnavis A., Commins C., Glynn C., Chun J.E., Perbet R, & Hyman B.T. (2023). Tau seeding and spreading in vivo is supported by both AD-derived fibrillar and oligomeric tau. Acta Neuropathologica, 146(2), 191-210.

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Immunofluorescence staining of cellular markers

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Following fixation with 4% paraformaldehyde for 10 min, cells were washed 3 times for 5 min with 0.1 M phosphate buffer (0.1 M Na₂HPO₄ × 2H₂O, 0.1 M NaH₂PO₄ × H₂O, pH 7.4) and blocked with 3% bovine serum albumin and 0.1% Triton X-100 in 0.1 M phosphate buffer for 30 min. After incubation at 4 °C overnight with 1:50 rabbit α-galectin-1 (Abcam, Cambridge, UK), 1:100 mouse α-smooth muscle-α-actin (Santa Cruz, Dallas, TX, USA), 1:100 mouse α-N-cadherin (Thermo Fisher), 1:100 mouse α-integrin-β1 (Merck), 1:100 mouse α-cytokeratin-8 (Merck), 1:100 phalloidin Alexa Fluor 555 (Thermo Fisher) in 0.3% bovine serum albumin, and 0.01% Triton X-100 in 0.1 M phosphate buffer, specimens were washed again 3 times for 10 min each with 0.1 M phosphate buffer and incubated with 1:1000 goat anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 488 (both from Thermo Fisher) in 1:10 diluted blocking solution for 1 h at room temperature. After nuclear staining with Hoechst 33342 (Thermo Fisher), specimens were washed again 3 times with 0.1 M phosphate buffer and mounted with the ProLong glass antifade mounting medium (Thermo Fisher). Immunofluorescence staining was analyzed by an Axio Observer 7 fluorescence microscope with an Apotome 2 module (Zeiss) and documented using the ZEN software Blue edition 3.0 (Zeiss).

Liesenhoff C., Paulus S.M., Havertz C., Geerlof A., Priglinger S., Priglinger C.S, & Ohlmann A. (2023). Endogenous Galectin-1 Modulates Cell Biological Properties of Immortalized Retinal Pigment Epithelial Cells In Vitro. International Journal of Molecular Sciences, 24(16), 12635.

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Micronuclei Quantification in Mitotic Cells

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3 × 10⁶ AC16 cells were plated in a 100-mm dish and treated with 100 ng/mL of nocodazole for 6h. Mitotic cells were harvested by shake-off and washed 3x with PBS. Mitotic cells were then counted and plated in on poly-L-ornithine (PLO)-coated #1.5 12 mm glass-coverslips (Thomas Scientific). Simultaneously, untreated AC16s were plated on PLO-coated coverslips and transfected with 40 nM of non-targeting dsiRNA or dsiRNA targeting MEF2A for 24 h. Cells were fixed with 4% Paraformaldehyde for 15 min at room temperature. Fixed cells were then washed 2 times with PBS and blocked in PBS containing 3% BSA and 0.3% Triton X-100. Cells were stained using Lamin B1 antibodies (CST) at 1:200 dilution for 1h at room temperature in PBS containing 1% BSA and 0.1% Triton X-100. Cells were washed 3 times and stained with Alexa 488 conjugated anti-rabbit secondary (Invitrogen) and DAPI (Thermo Scientific) for 1h at room temperature (RT) in PBS containing 1% BSA and 0.1% Triton X-100. Cells were washed and mounted with ProLong Glass Antifade mounting medium (Thermo Fisher). Samples were imaged using a Nikon Eclipse Ti laser scanning confocal microscope, 60x oil-immersion lens. Percent micronuclei was quantified by counting the number of micronuclei per nucleus in the field of view using FIJI.^{74 (link)}

Pohl T., Zimmer M., Mugele K, & Spiess J. (1991). Primary structure and functional expression of a glutaminyl cyclase.. Proceedings of the National Academy of Sciences of the United States of America, 88(22), 10059-10063.

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Analyze B16F10 Rnf31-KO Cell Response

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B16F10 Rnf31-KO Cells (2.5×10⁶) were plated on a poly-D-Lysine (Gibco) coated glass slides placed in a 100 mm-dish in 10ml of complete DMEM. Cells were treated for 4.5h with TNFα (100ng/ml), fixed with ice-cold methanol for 15 minutes at −20°C, washed twice with PBS, and blocked with 3% rat serum in PBS for 15 minutes at 4°C. Cells were then stained with primary antibodies for 15 minutes at 4°C, washed twice with 3% rat serum in PBS, stained with secondary antibodies and DAPI (25 μg/ml, Life Technologies) for 15 minutes at 4°C, and washed 3 times with 3% rat serum in PBS. Primary and secondary antibodies were diluted in blocking buffer. Coverslips were mounted using Prolong Glass Antifade Mounting Medium (ThermoFisher). Imaging was performed using a Zeiss LSM 980 confocal microscope. Images were captured using Zen Software. Three independent experiments with three biological replicates per group were performed. For cleaved caspase 8 detection, anti-cleaved caspase 8 rabbit mAb (1:200 dilution) (clone D5B2, Cell Signaling, #8592) and secondary antibody Alexa Fluor 647-goat anti-rabbit IgG (5 μg/ml, Southern Biotech) were used. Alexa Fluor 488 anti-LC3B antibody (1:100 dilution) (clone EPR18709, Abcam, #ab225382) was used to detect LC3B.

Ito Y., Pan D., Zhang W., Zhang X., Juan T.Y., Pyrdol J.W., Kyrysyuk O., Doench J.G., Liu X.S, & Wucherpfennig K.W. (2023). Addressing Tumor Heterogeneity by Sensitizing Resistant Cancer Cells to T cell-secreted Cytokines. Cancer discovery, 13(5), 1186-1209.

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