Slowfade mounting media

Manufactured by Thermo Fisher Scientific

Sourced in United States

Slowfade mounting media is a product designed for use in fluorescence microscopy. It is formulated to minimize the fading of fluorescent signals over time, helping to preserve the quality of fluorescent images during observation and analysis.

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Lab products found in correlation

14 protocols using slowfade mounting media

Immunofluorescence Staining of Patiria miniata Embryos

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Patiria miniata embryos were fixed in 4% paraformaldehyde/PBS for 20 min at RT, followed by permeabilization in 1% Triton X-100/PBS for 10 min. Embryos were then washed four times in PBS/0.1% Triton X-100 and post-fixed in ice cold methanol for 20 min. After another four washes, embryos were blocked in 3% BSA/PBS for 30 min and incubated with anti-PmTbr (1:500) overnight at 4 °C. Affinity purified polyclonal anti-PmTbr was produced in rabbits by Piece Custom Antibody Services. Embryos were washed four times and incubated in 1:100 FITC anti-rabbit (Sigma) overnight. Embryos were incubated in 1:10,000 DAPI (Life Technologies) for 30 min, washed four times in PBS/0.1% Triton X-100.
Embryos were imaged in Slowfade mounting media (Life Technologies) by confocal microscopy.

Cheatle Jarvela A.M., Brubaker L., Vedenko A., Gupta A., Armitage B.A., Bulyk M.L, & Hinman V.F. (2014). Modular Evolution of DNA-Binding Preference of a Tbrain Transcription Factor Provides a Mechanism for Modifying Gene Regulatory Networks. Molecular Biology and Evolution, 31(10), 2672-2688.

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Fluorescent Membrane Dye and Mounting Media Protocol

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Fast DiO, 3,3′-Dilinoleyloxacarbocyanine Perchlorate, fluorescent membrane dye and Slow Fade Mounting media were obtained from Life Technologies (Carlsbad, CA). Cell culture media and supplements, Fetal Bovine Serum (FBS), Phosphate Saline Buffer (PBS), Trypsin, and antibiotics were from Fisher Scientific International, Inc. (Hampton, NH). MicroBCA Protein Assay kits were from Thermo Fisher Scientific Inc. (Waltham, MA). All blocking agents including Bafilomycin A1, Nystatin and Chlorpromazine were purchased from Sigma-Aldrich (St. Louis, MO).

Banizs A.B., Huang T., Nakamoto R.K., Shi W, & He J. (2018). Endocytosis pathways of endothelial cell derived exosomes. Molecular pharmaceutics, 15(12), 5585-5590.

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Immunofluorescent Staining of Retinal Cells

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Eyes were enucleated under anesthesia before euthanasia. Enucleated eyes were incubated in PBS for 10 minutes at RT and then in 4% paraformaldehyde/PBS for 15 minutes on ice. Retinas were removed carefully with Vanna’s scissors and fine forceps, washed with 0.1% Tween 20/Tris-buffered saline (TBS), and blocked with 2% normal goat serum/TBS for 2 hours. The retinas were stained with anti-mouse antibodies CD4-FITC and FoxP3-eFluor 570 (eBioScience) and Lectin-AlexaFluor647 (Life Technologies) overnight and then with DAPI (Invitrogen, Life Technologies) for 15 minutes, followed by mounting with Slowfade mounting media (Life Technologies). Confocal z-stacked images were obtained through an Olympus 7000 (Center Valley, PA, USA). The confocal images were converted to single channel split images before counting CD4-FITC+, FoxP3-eFluor 570+ or CD4-FITC+, FoxP3-eFluor 570- cells using ImageJ (NIH, Bethesda, MD, USA).

Nakamura Y.K., Janowitz C., Metea C., Asquith M., Karstens L., Rosenbaum J.T, & Lin P. (2017). Short chain fatty acids ameliorate immune-mediated uveitis partially by altering migration of lymphocytes from the intestine. Scientific Reports, 7, 11745.

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Immunostaining of Transfected Cells

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In an alternative immunostaining experiments, 3–5 days post-transfection cells were fixed in 4% Paraformaldehyde at room temperature for 20 min. Cells were then rinsed three times with 1X PBS, then 5 min in 50 mM NH4Cl, and three more quick rinses in 1X PBS. Cells were permeabilized in 0.1% Tx-100 PBS for 5 min followed by three quick PBS rinses. Cells were incubated in freshly made 0.5% Sodium Borohydrate in PBS for 5 min. Cells were quickly rinse in PBS and incubated in 2 mL of 1% BSA in PBS for 45 min followed by incubation in 100 μL of anti-Pin1 (1:500) and anti-PSD-95 (1:500) for 1 h or overnight at 4°C (Table 1). Cells were rinsed 3X in PBS and the Alexa fluor 647 anti-mouse (1:500), and Alexa fluor 488 anti-rabbit applied at a dilution of 1:500 for 1 h in 1% BSA in PBS. Cells were rinsed 5X in PBS, post fixed in 4% PFA and mounted in slow fade mounting media (Life technologies).

Delgado J.Y., Nall D, & Selvin P.R. (2020). Pin1 Binding to Phosphorylated PSD-95 Regulates the Number of Functional Excitatory Synapses. Frontiers in Molecular Neuroscience, 13, 10.

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Confocal Imaging of Lymph Node Tissues

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Confocal imaging was done using standard conditions. In brief, LN were excised, fixed for 2 hours at room temperature in 4% paraformaldehyde, and dehydrated at 4°C in 30% sucrose in PBS. Tissues were snap frozen in OCT compound (Sakura Tissue-Tek). 10 μm tissue sections were cut, fixed in acetone for 20 minutes at −20°C and dried overnight. Tissue sections were rehydrated in PBS for 10 minutes prior to staining. Staining was done in 10% donkey serum (Jackson ImmunoResearch) 0.3% Triton X-100 in 1x PBS overnight. Tissues were imaged in Slow Fade mounting media (Life Technologies). All images were acquired using LSM880 (Carl Zeiss) or SP8 (Leica) confocal microscopes with a 40X oil immersion objective. Images were processed and analyzed using ImageJ software (version 2.0.0-rc-54/1.51h; National Institutes of Health).

Mendoza A., Yewdell W.T., Hoyos B., Schizas M., Bou-Puerto R., Michaels A.J., Brown C.C., Chaudhuri J, & Rudensky A.Y. (2021). Assembly of a spatial circuit of T-bet-expressing T and B lymphocytes is required for antiviral humoral immunity.. Science immunology, 6(60), eabi4710.

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Retinal Immune Cell Quantification

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Enucleated eyes were incubated in 4% paraformaldehyde in PBS for 30 minutes on ice, and retinas were dissected from the surrounding tissue, washed in Tris-buffered saline, followed by Tris-buffered saline with Triton X-100. The retinas were then blocked with normal goat serum–containing buffer for 2 hours, and then stained overnight with CD4-FITC, Foxp3-efluor570 (both eBioscience), and Lectin-AlexaFluor647 (Life Technologies). Retinas were then washed, stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 minutes, and mounted in Slowfade mounting media (Life Technologies). An Olympus 7000 (Center Valley, PA, USA) was used to acquire images, and ImageJ (ImageJ.nih.gov/ij/">http://ImageJ.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) was used to convert z-stacked images to dual color images for counting of CD4-FITC/Foxp3-eFluor570 double positive cells.

Nakamura Y.K., Metea C., Karstens L., Asquith M., Gruner H., Moscibrocki C., Lee I., Brislawn C.J., Jansson J.K., Rosenbaum J.T, & Lin P. (2016). Gut Microbial Alterations Associated With Protection From Autoimmune Uveitis. Investigative Ophthalmology & Visual Science, 57(8), 3747-3758.

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Immunofluorescence Staining of Pin1 and Phospho-T19

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Three to five days post-transfection cells were fixed in 4% Paraformaldehyde at room temperature for 20 min. Cells were then rinsed three times with 1× PBS, then 5 min in 50 mM NH4Cl, and three more quick rinses in 1× PBS. Cells were permeabilized in 0.1% Tx100 PBS for 5 min followed by three quick PBS rinses and incubated in 2 mL of 1%BSA in PBS for 45 min followed by incubation in 100 μl of anti-Pin1 (Santa Cruz Biotechnology, Dallas, TX, USA) and the anti-phospho T19 (1:500) for 1 h. Cells were rinsed 3× in PBS and the Alexa fluor 647 anti-mouse (1:500), and Alexa fluor 488 anti-rabbit (1:500) for 1 h in 1% BSA in PBS. Cells were rinsed 5× in PBS, postfix in 4% PFA and mounted in slow fade mounting media (Life Technologies, Carlsbad, CA, USA).

, & Delgado J.Y. (2020). An Alternative Pin1 Binding and Isomerization Site in the N-Terminus Domain of PSD-95. Frontiers in Molecular Neuroscience, 13, 31.

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Immunohistochemical Staining of Mouse Brain

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Mice were deeply anesthetized with sodium pentobarbital (Euthasol; 0.39 mg drug/gm body weight; 150mg/kg; APP Pharmaceuticals; Schaumburg, IL) and perfused intracardially with 0.01M phosphate buffered saline (PBS; pH 7.4) followed by 4% buffered formaldehyde. Brains were post-fixed for at least 2 hrs. Sixty μm coronal vibratome sections were rinsed four times for five minutes in PBS, and then incubated in 0.01 M citrate buffer (pH 8.5) at 80°C for 25 minutes (Jiao et al., 1999 (link)). Tissue was cooled to room temperature, washed twice in PBS for three minutes, and permeabilized in 0.03% Triton in PBS (TW). Sections were then placed into a blocking solution (0.5% normal donkey serum in TW; Jackson ImmunoResearch, West Grove, PA) for one hr, followed by overnight incubation in primary antibody (Table 1) at 4°C. Sections were then rinsed four times in PBS, and placed in a secondary antibody solution (1/250 to 1/500 in TW: Jackson ImmunoResearch, donkey anti-rabbit: Catalog number, 711-165- 152 or 711-545-152; donkey anti-goat: 705-165- 147 or 705-545- 147; donkey anti-mouse: 715-485- 150 or 715-545- 151) for 2 hrs. Tissue went through a final 4×5min PBS rinse before being mounted on slides with SlowFade mounting media (Invitrogen: S36937)

Collins L.N., Hill D.L, & Brunjes P.C. (2018). Myelination of the developing lateral olfactory tract and anterior commissure. The Journal of comparative neurology, 526(11), 1843-1858.

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Claudin-1 and ZO-1 Localization in Caco-2 Cells

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For in vitro cell culture experiments, Caco-2 cells were treated with IL-1β for 24 hr and washed twice with PBS (Invitrogen) and fixed for 15 min with an acetone/methanol solution (1/1 v/v), permeabilized with a 1% Triton X-100 solution (Sigma-Aldrich) and blocked with 3% milk in PBS for 1 hr. Cells were then incubated with primary antibodies (Claudin-1, and ZO-1) overnight at 4°C followed by an incubation with a secondary antibody (1∶200) for 1 hr. Filters were mounted on slides with coverslip using Slow fade mounting media (Invitrogen). Slide were then analysed using a LSM 700 microscope (Zeiss) and fluorescence level was quantified using Image J software.

Hirao L.A., Grishina I., Bourry O., Hu W.K., Somrit M., Sankaran-Walters S., Gaulke C.A., Fenton A.N., Li J.A., Crawford R.W., Chuang F., Tarara R., Marco M.L., Bäumler A.J., Cheng H, & Dandekar S. (2014). Early Mucosal Sensing of SIV Infection by Paneth Cells Induces IL-1β Production and Initiates Gut Epithelial Disruption. PLoS Pathogens, 10(8), e1004311.

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Immunofluorescence Staining of Cultured Cells

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Cultured cells were fixed with 4% PFA for 15 minutes at room temperature (RT) and block-permeablised using PBST-10% Normal Donkey Serum (NDS) for 1 hour at RT. Primary and secondary antibodies were incubated in 3%NDS overnight (O/N) at 4 °C and 1 hour at RT respectively at the following dilutions; mouse anti-CNPAse, (1:2000; Chemicon, Germany), rabbit anti-GFAP, goat anti-GFAP, mouse anti-βIII-tubulin, rabbit anti-βIII-tubulin (all at 1:300; Sigma-Aldrich), rabbit anti-Pax6 (1:200; Chemicon, Germany), donkey anti-sheep Alexafluor555, donkey anti-rabbit Alexafluor488/555/647 and donkey anti-mouse Alexafluor488/555/647 (all 1:1000; Invitrogen), donkey anti-chickenCy3 (Jackson Laboratories, Bar Harbour, ME). Cells were counterstained with DAPI and mounted with Slow-fade mounting media (Invitrogen). Non-specific staining was controlled by using secondary-only controls. Fluorescence was viewed using the Axioplan2 microscope (Carl Zeiss, Jena, Germany) fitted with an HBO 100 lamp (Carl Zeiss, Jena, Germany). Images were captured using an Axiocam Mrm camera and Axio Vs40 v4.5.0.0 software (Axiovision, Carl Zeiss, Jena, Germany).

Bridges C.R., Tan M.C., Premarathne S., Nanayakkara D., Bellette B., Zencak D., Domingo D., Gecz J., Murtaza M., Jolly L.A, & Wood S.A. (2017). USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors. Scientific Reports, 7, 391.

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