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Antifade reagent

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Antifade reagent is a liquid solution designed to protect fluorescent dyes from photobleaching during microscopy and imaging applications. It helps maintain the brightness and stability of fluorescent signals over time.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions) Prolong gold mounting medium, by Thermo Fisher Scientific (4 mentions) Cx41 fluorescence microscope, by Olympus (3 mentions) Dmi6000b, by Leica (3 mentions) Paraformaldehyde (pfa), by Merck Group (3 mentions) Lsm 900 confocal microscope, by Zeiss (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions) Alexa fluor 488 donkey anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions) Ds ri1 microscope, by Nikon (2 mentions) Ab189841, by Abcam (2 mentions) Dfc7000t, by Leica (2 mentions) Hoechst 33342 dye, by Thermo Fisher Scientific (2 mentions) Methanol, by Merck Group (2 mentions) Alexa fluor 594, by Thermo Fisher Scientific (2 mentions) Tcs sp8 confocal microscope, by Leica (2 mentions) Triton x 100, by Merck Group (2 mentions) Poly l lysine (pll), by Merck Group (2 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Opal multiplex tissue staining kit, by PerkinElmer (2 mentions)

Close spelling variants of this product

ProLong Gold antifade reagent with DAPI (1478 citations) ProLong Diamond antifade reagent (109 citations) ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (60 citations) SlowFade Antifade reagent (32 citations) Antifade reagent containing DAPI (11 citations) Antifade mounting reagent (8 citations) ProLong (R) Gold antifade reagent (7 citations) ProLong® Gold Antifade Mountant reagent (6 citations) ProLong Gold antifade reagents with DAPI (5 citations) ProLong Diamond Antifade Mountant with DAPI reagent (5 citations)

99 protocols using antifade reagent

1

Immunofluorescence Staining of Cultured Neurons and Brain Tissue

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Cultured neurons on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, blocked for 1 h in blocking solution (Roche) with 0.3% TritonX-100 in PBS, and incubated in diluted primary antibody solution overnight at 4 °C. Cells were washed the next day in PBS and incubated in diluted secondary antibodies for 2 h before the final washing. Coverslips were mounted with antifade reagent (Thermo Fisher Scientific).
Embryonic brains and perfused mouse post-natal (14 days) brains were fixed with 4% paraformaldehyde in PBS overnight at 4 °C, and then transferred to 20% sucrose in PBS overnight and changed to 30% sucrose in PBS overnight at 4 °C. Brains were embedded in O.C.T compound (Sakura). Brains were cryosectioned into 50 μm thick slices. Brain slices were washed 3 times in PBS and incubated in diluted Hoechst (Thermo Fisher Scientific) solution for 2 h before the final washing. After nuclei staining, brain slices were mounted with antifade reagent (Thermo Fisher Scientific).
Kim J.Y., Hwang H.G., Lee J.Y., Kim M, & Kim J.Y. (2020). Cortactin deacetylation by HDAC6 and SIRT2 regulates neuronal migration and dendrite morphogenesis during cerebral cortex development. Molecular Brain, 13, 105.
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2

Receptor Distribution Analysis in Cells

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Cells were fixed with 3.7% formaldehyde for 20 min and permeabilized in 0.2% Triton X-100 for 4 min. For receptor distribution studies, cells were labeled with anti-CXCR4 (1:100), anti-β1 integrin (1:400), or anti-CD44 (1:100) antibodies. Primary antibodies were detected using Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 568-conjugated goat anti-mouse, or Alexa Fluor 647-conjugated goat anti-mouse antibodies (at 1:200, Molecular Probes, Invitrogen, France). All the slides were mounted onto coverslips with antifade reagent (Prolong, Invitrogen). Observations were made on an Axiovert 200 M microscope and a LSM 700 confocal microscope (both from Carl Zeiss SAS, Le Pecq, France) using 20X or 63X objectives. Images were acquired with Metaview software using a CoolSNAP EZ CCD camera (both from Ropper Scientific, Evry, France).
Liu X.Q., Fourel L., Dalonneau F., Sadir R., Leal S., Lortat-Jacob H., Weidenhaupt M., Albiges-Rizo C, & Picart C. (2017). Biomaterial-enabled delivery of SDF-1α at the ventral side of breast cancer cells reveals a crosstalk between cell receptors to promote the invasive phenotype. Biomaterials, 127, 61-74.
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3

Visualizing Autophagy and Lysosomal Dynamics

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The HeyA8-MDR and OV2008 (EV, CTSL+/+) cells were grown on multi-chambered slides overnight. HeyA8-MDR cells were treated with QC at 0 and 5 µM for 24 h and OV2008 cells were exposed to Magic Red® and then cells were fixed with 100% methanol. After blocking with 1% BSA in PBS, the cells were incubated at room temperature for 1 h with respective antibodies to p62 or CTSL and then washed three times with 1X PBS. p62 was detected using rabbit anti-mouse IgG Alexa Fluor® 594 or rabbit anti-mouse IgG-FITC (Molecular Probes, Eugene, OR, USA and CTSL with Alexa Fluor® 488 donkey anti-goat IgG or Magic Red® in 1% BSA. C13, HeyA8-MDR, OV2008 (EV, CTSL+/+), C13 (NTC, CTSL-sh1) cells were transfected transiently with GFP-RFP-LC3/or p62 plasmid and treated with or without QC for validating autophagic flux. Slides were mounted with Antifade reagent (Invitrogen, Carlsbad, CA, USA), visualized using a Zeiss-LSM 510 microscope and corrected total cell fluorescence (CTCF) was obtained by ImageJ-Fiji software.
Thirusangu P., Pathoulas C.L., Ray U., Xiao Y., Staub J., Jin L., Khurana A, & Shridhar V. (2021). Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death. Cancers, 13(9), 2004.
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4

Visualizing MHV68 Infection in RL-65 Cells

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RL-65 cells were plated at a concentration of 2×105 onto 1.7 cm2 glass chamberslides (Millipore). After visual confirmation of confluency, cells were infected at a MOI of 0.05 with MHV68-H2bYFP, a virus that expresses the yellow fluorescent protein from infected cells [35] (link). 120 hours post infection cells were fixed with 3.7% formaldehyde for 20 minutes at room temperature and mounted with antifade reagent containing DAPI (Invitrogen) and visualized for YFP expression.
Scott F.M, & Speck S.H. (2014). A Tissue Culture Model of Murine Gammaherpesvirus Replication Reveals Roles for the Viral Cyclin in Both Virus Replication and Egress from Infected Cells. PLoS ONE, 9(4), e93871.
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5

Histological Staining and Imaging Techniques

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For H&E staining, tissues were fixed in 4% paraformaldehyde overnight at room temperature (RT) and followed by dehydration in 70% ethanol. tissues were embedded in paraffin, sectioned at a thickness of 5 μm, and stained with H&E following the standard protocol. COS-7 cells transfected with CSF1R-GFP were washed in PBS and incubated with parvalbumin (200 ng/ml) for 30 min on ice. Sodium azide and Pitstop 2 (ab120687) were added to block receptor internalization. Cells were then fixed with 4% paraformaldehyde for 15 min and blocked in 5% BSA for 30 min at RT and subsequently incubated with parvalbumin antibody (1:1000) in 5% BSA for 2 h at RT. After incubating with a secondary antibody against rabbit IgG conjugated to CY3, cells were co-stained with DAPI (Sigma), and mounted with Antifade reagent (Invitrogen) for fluorescence microscopy. For oil red o staining, tissue slices fixed with 4% paraformaldehyde and embedded with OCT (TissueTek) were rapidly frozen using liquid nitrogen and stored at −80 °C. Sections in 10 μm thick were incubated with 0.3% oil red O solution (in 60% isopropanol) at RT for 10 min and followed by microscope analysis. Images were captured using a Leica DFC 5400 digital camera and were processed using Leica Application Suite v4.13
Lin S., Zhang A., Yuan L., Wang Y., Zhang C., Jiang J., Xu H., Yuan H., Yao H., Zhang Q., Zhang Y., Lou M., Wang P., Zhang Z.N, & Luan B. (2022). Targeting parvalbumin promotes M2 macrophage polarization and energy expenditure in mice. Nature Communications, 13, 3301.
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6

Quantification of Stress Granule Size in Mammalian Cells

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Cells were passaged to a 6-well plate with a coverslip in each well and cultured overnight. The cells were washed once in PBS and then fixed using 4% paraformaldehyde in PBST (PBS with 0.05% Tween-20) at room temperature for 15 min. Then, the cells were washed twice by PBST and permeabilized by 0.5% Triton at room temperature for 20 min. After being washed once by PBST, the cells were blocked with 1% BSA in PBST at room temperature for 30 min. Then, the blocking solution was replaced with 1 mL blocking solution supplemented with desired primary antibodies (at 1:1000 dilution) and incubated at room temperature for 1 hr or 4 °C overnight. After 4 washes with PBST, the corresponding Alexa Fluor-conjugated secondary antibodies were applied (1:1,000 diluted in the blocking solution) and incubated at room temperature for 1 hr. After being washed three times by PBST, the cells were incubated with 0.5 μg/mL DAPI for 1 min. After 4 times PBST washes, an antifade reagent (Invitrogen) was used to mount the slides. The images were taken using a Leica TCS SP8 confocal microscope. The “analyze particles tool” in Fiji (Schindelin et al., 2012 (link)) was utilized to quantify the sizes of the stress granules in mammalian cells in about 50 different cells per condition. The sizes of the stress granule in cells were analyzed by two researchers independently.
Shen H., Yanas A., Owens M.C., Zhang C., Fritsch C., Fare C.M., Copley K.E., Shorter J., Goldman Y.E, & Liu K.F. (2022). Sexually dimorphic RNA helicases DDX3X and DDX3Y differentially regulate RNA metabolism through phase separation. Molecular cell, 82(14), 2588-2603.e9.
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7

Assessing Testis Histology and 5hmC Levels

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To analyse testis, part of the tissue was fixed in Bouin's solution overnight. The fixed tissues were then embedded into paraffin, and 5-m sections were cut with a Microtome. After being deparaffinized, and rehydrated, sections were stained with hematoxylin-eosin. For 5hmC staining, post-implantation embryos were dissected from pregnant females at the day indicated. Then embryos were fixed with 4% paraformaldehyde, embedded in OCT compound (Sakura) and 8 μm sections were cut. After washing with PBS, the sections were treated with hydrochloric acid solution (4N hydrochloric acid, 0.1% Triton X-100 in distilled water), followed by washing with PBS and permeabilization with blocking buffer (1% BSA, 10% goat serum, PBS containing 0.3% Triton X-100). Next, sections were incubated with primary antibodies (mouse anti-5mC antibody, 1:500, Eurogentec#BI-MECY-0100; rabbit anti-5hmC antibody, 1:1 000, Active Motif #39792) overnight at 4 °C and secondary antibodies at room temperature for 1 h. Finally, the slides were mounted in Anti-fade Reagent (Invitrogen) and imaged using a LEICA TCS SP5 II confocal microscope. The signal intensity was determined using Leica Application Suite software. Note, for embryos with Tet1, 2, 3 triple KO, we used the background (non-specific) for 5hmC to calculate the average ratio of 5hmC/5mC.
Zuo E., Cai Y.J., Li K., Wei Y., Wang B.A., Sun Y., Liu Z., Liu J., Hu X., Wei W., Huo X., Shi L., Tang C., Liang D., Wang Y., Nie Y.H., Zhang C.C., Yao X., Wang X., Zhou C., Ying W., Wang Q., Chen R.C., Shen Q., Xu G.L., Li J., Sun Q., Xiong Z.Q, & Yang H. (2017). One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Research, 27(7), 933-945.
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8

Imaging Cellular Uptake of siRNA-Loaded Nanoparticles

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Cellular uptake of the nanoparticles loaded with the AF488-conjugated siRNA was imaged by fluorescence microscopy. The macrophages were seeded on cover slide in 6-well culture plate at 1 × 106 cells/well and treated by the nanoparticles loaded with 5 μg of the AF488-conjugated siRNA. For intracellular localization of the AF488-MIF-siRNA, the macrophages were incubated with lysotracker for 30 minutes before the treatment with the BG34-10-Re-I/(AF488-MIF-siRNA) nanoparticles. After the treatment, the macrophages were washed by PBS and fixed by 1% paraformaldehyde. The fixed macrophages were incubated with Hoechst blue 33342 for 15 minutes and mounted on a glass slide using mounting and anti-fade reagent from Invitrogen. The fluorescence images were acquired on a Carl Zeiss fluorescent microscope system LSM 510 and processed by Zeiss LSM Image software (Zeiss, Jena, Germany).
Zhang M., Gao Y., Caja K., Zhao B, & Kim J.A. (2015). Non-Viral Nanoparticle Delivers Small Interfering RNA to Macrophages In Vitro and In Vivo. PLoS ONE, 10(3), e0118472.
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9

Quantifying H4K12 Acetylation in Oocytes

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To measure the H4K12 acetylation levels, we collected oocytes from the OGCs at the end of the culture period (14 days). We fixed the oocytes in 4% paraformaldehyde, incubated them with a
primary antibody (rabbit polyclonal anti- histone H4 acetyl K12, 1:200; Abcam, Cambridge, UK), then incubated them with a secondary antibody (fluorescein-conjugated goat anti-rabbit IgG,
1:500; Cell Signaling Technology, Danvers, MA). The oocytes were then mounted on slides with an anti-fade reagent containing DAPI (Invitrogen). Images of oocytes were captured under a
fluorescence microscope (Keyence). We quantified the fluorescence intensities of the whole oocytes using ImageJ software (NIH, Bethesda, MD, USA).
MUNAKATA Y., SUGIMOTO A., SHIRASUNA K., KUWAYAMA T, & IWATA H. (2019). Xanthan gum and Locust bean gum gel supports in vitro development of porcine oocytes derived from early antral follicles. The Journal of Reproduction and Development, 65(6), 551-554.
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10

Immunostaining of Endogenous Nuclear REST

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Cells plated on laminin/polylysine-coated coverslips were fixed by incubation with 4% (v/v) paraformaldehyde in PBS for 30 minutes at room temperature, followed by three washes in PBS, and then permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature. After 3 additional washes in PBS, cells were blocked with 10% goat serum in PBS for 1 hour at room temperature. Primary antibodies were diluted to the appropriate concentration using 10% goat serum, and incubated overnight at 4°C. Cells were then washed 3 times in 1% goat serum in PBS for 10 minutes before adding fluorophore-conjugated secondary antibodies for 2 hours at room temperature. Fluorophore labeled cells were then washed in 1% goat serum in PBS for 3×10 minutes, and then mounted using Prolong Gold mounting medium with DAPI and anti-fade reagent (Invitrogen). To score endogenous nuclear REST in SH-SY5Ycells, Pearson correlations between DAPI and REST were generated from 200–300 cells from multiple coverslips using confocal analysis software. A threshold of R>0.6 was used to indicate positive nuclear localization.
Lu T., Aron L., Zullo J., Pan Y., Kim H., Chen Y., Yang T.H., Kim H.M., Drake D., Liu X.S., Bennett D.A., Colaiácovo M.P, & Yankner B.A. (2014). REST and Stress Resistance in Aging and Alzheimer’s Disease. Nature, 507(7493), 448-454.
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