To observe changes in the localization of protein fluorescent tags during microspore development, anthers of different stages were dissected and placed into SlowFade™ Diamond Antifade Mountant (Thermo Fisher Scientific), while gently pressing the sample with a coverslip to allow the dye to penetrate. The fluorescence signals of eYFP/EGFP/mCherry protein were captured using a Leica SP8 confocal microscope. More detailed observations of the stained tetrads were performed as previously described22 (link). Briefly, anthers from the tetrad stage were dissected, placed in SlowFade™ Diamond Antifade Mountant supplemented with 0.02% calcofluor white and 5 mg ml−1 membrane stain Cell Mask Deep Red (Molecular Probes), and then imaged using a confocal microscope. Calcofluor white fluorescence was imaged at an excitation wavelength of 405 nm and an emission wavelength of 424–475 nm, Cell Mask Deep Red fluorescence was imaged at an excitation wavelength of 640 nm and an emission wavelength of 663–738 nm, eYFP fluorescence was imaged at an excitation wavelength of 514 nm and an emission wavelength of 522 to 555 nm.
Slowfade diamond antifade mountant
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Australia
SlowFade Diamond Antifade Mountant is a specialized reagent designed to minimize photobleaching of fluorescent dyes during microscopy imaging. It is formulated to maintain the brightness and stability of fluorescent signals over time.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 confocal microscope, by Leica (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Las x software, by Leica (2 mentions)
Tcs sp8 confocal microscope, by Leica (2 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Alexa 488, by Jackson ImmunoResearch (2 mentions)
Millicell ez 8 well chamber slides, by Merck Group (2 mentions)
Tetramethylrhodamine conjugated phalloidin, by Thermo Fisher Scientific (2 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
Dp controller and dp manager software, by Olympus (2 mentions)
Dp71 fluorescent microscope, by Olympus (2 mentions)
Hoechst, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Cellmask deep red, by Thermo Fisher Scientific (2 mentions)
81 protocols using slowfade diamond antifade mountant
1
Fluorescent Protein Localization in Microspore Development
2
Quantifying Argyrophilic Nuclear Organizing Regions in Paraffin-Embedded Tumor Slides
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The paraffin-embedded tumor slides from mice were deparaffinized in xylene, hydrated using a graded series of alcohol, and assessed for argyrophilic nuclear organizing regions (AgNOR) according to a modified protocol (37 (link)). Cells were fixed in 2% glutaraldehyde followed by postfixation in ethanol–acetic acid solution (3:1), stained using a 0.33% formic acid–33.3% silver nitrate solution in 0.66% gelatin, and then mounted on slides using SlowFade Diamond Antifade Mountant (Invitrogen). AgNOR numbers in at least 100 cells on each slide were quantified at 100× magnification. Experiments were repeated three times.
3
Immunohistochemical and Histochemical Analysis of Amyloid-beta Plaques in Brain Sections
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30 µm thick free-floating sagittal brain sections were washed 3 × 10 min in TBS, treated 8 min with 88% formic acid (FA), permeabilized 3 × 10 min in TBS containing 0.25% Triton X-100 (TBSX), and blocked 1 h in TBSX containing 5% normal donkey serum (NDS). The sections were then incubated with anti-Aβ42 primary antibody (H31L21, Invitrogen) diluted 1:1000 in TBSX containing 2.5% NDS overnight at 4 °C. Following overnight incubation, the sections were washed 3 × 10 min in TBSX, incubated with appropriate Alexa-fluorophore-conjugated secondary antibody diluted 1:500 in TBSX containing 2.5% NDS, washed 3 × 10 min in TBSX, mounted on glass slides, and coverslipped with ProLong™ Diamond Antifade Mountant (Invitrogen) according to the recommendations from the manufacturer.
For the detection of fibrillar dense-core plaques, free-floating sagittal sections were stained with 0.01% thioflavin S in 50% ethanol for 10 min and then washed 2 × 1 min in 50% ethanol, 3 × 1 min in ddH2O, and finally 10 min in TBS. The stained specimens were mounted on glass slides and coverslipped with SlowFade™ Diamond Antifade Mountant (Invitrogen) according to the manufacturer’s recommendations.
For the detection of fibrillar dense-core plaques, free-floating sagittal sections were stained with 0.01% thioflavin S in 50% ethanol for 10 min and then washed 2 × 1 min in 50% ethanol, 3 × 1 min in ddH2O, and finally 10 min in TBS. The stained specimens were mounted on glass slides and coverslipped with SlowFade™ Diamond Antifade Mountant (Invitrogen) according to the manufacturer’s recommendations.
4
Immunostaining of Intestinal ACE2 and Cytokeratin
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Transmural ileal biopsies, obtained during abdominal surgery in patients with IBD and CRC, were fixed in 4% formalin and embedded in paraffin, and sections of 5 µm were cut [Translational Cell & Tissue Research Laboratory, University Hospitals Leuven, and at VIB & KU Leuven Center for Brain & Disease Research]. After deparaffinisation, antigen retrieval was done in Tris-EDTA buffer [10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0] at 95°C for 30 min; 1% BSA in PBST [0.1% Tween-20 and 0.5% sodium azide] was used to block non-specific binding of detection antibodies and gently permeabilise before ACE2 and Cytokeratin AE1/AE3 staining. In brief, ACE2 [Polyclonal, Cell Signaling Technology] and cytokeratin [IgG1-kappa, clone AE1/AE3, Dako] were applied in 1% BSA, followed by donkey anti-rabbit Cy3 [Jackson Immuno Research] and donkey anti-mouse Alexa fluor 488 [Invitrogen]. Slides were mounted in SlowFade™ Diamond Antifade Mountant [Invitrogen], and stored at 4 °C before imaging. Images were acquired using a Zeiss LSM 780 at the Cell and Tissue Imaging Cluster [CIC] at KU Leuven.
5
AgNOR Quantification in Paraffin-Embedded Tumor Slides
6
Mosquito Sensory Appendage Imaging
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Female mosquitoes (≥ 7 dpe) were briefly chilled on ice. Individual sensory appendages were removed with a pair of forceps or a fine microblade and mounted on microscope slides in a drop of mounting solution (SlowFade Diamond Antifade Mountant; Invitrogen, S36972). Legs were mounted directly after being detached from the bodies. To minimize air bubbles, antennae, palps, and labella were incubated in a fixative solution (4% paraformaldehyde in 0.1 M Millonig’s phosphate buffered solution) for 5 minutes prior to being mounted on slides. Imaging of sensory appendages was done within 2 hours of detaching from the body to capture the endogenous GFP signal.
Images of brains, VNCs, and sensory appendages were obtained using a Zeiss LSM 700 confocal microscope equipped with Fluar 10×/0.50 air M27, LCI Plan-Neofluar 25×/0.8 water Korr DIC M27, and Plan-Apochromat 40×/1.3 Oil DIC M27 objectives. Images were acquired at 1024 × 1024-pixel resolution. Images of brains and VNCs were captured with 2.45 μm z-steps using 25x objective. For sensory appendages imaged with 10x and 40x objectives, the z-steps were 2.84 μm and 0.42 μm, respectively. Maximum intensity projections of full z-stacks or partial z-stacks were generated using Fiji/ImageJ (https://fiji.sc/ ).
Images of brains, VNCs, and sensory appendages were obtained using a Zeiss LSM 700 confocal microscope equipped with Fluar 10×/0.50 air M27, LCI Plan-Neofluar 25×/0.8 water Korr DIC M27, and Plan-Apochromat 40×/1.3 Oil DIC M27 objectives. Images were acquired at 1024 × 1024-pixel resolution. Images of brains and VNCs were captured with 2.45 μm z-steps using 25x objective. For sensory appendages imaged with 10x and 40x objectives, the z-steps were 2.84 μm and 0.42 μm, respectively. Maximum intensity projections of full z-stacks or partial z-stacks were generated using Fiji/ImageJ (
7
Cytoskeleton Analysis of Drug-Treated Cells
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B35 or C6 cells were cultured with MK-801 for 7 days and then incubated with MK-801 in combination with or without PRP/WP for another 5 days. 5 × 103 drug-treated B35 or C6 cells were then seeded in 6-well plate coverslips coated with poly-L-lysine. The cells were further cultured for another 2 days in medium with corresponding MK-801, PRP, or WP. The coverslips with drug-treated B35 or C6 cells were moved to a new 6-well plate and fixed by incubating the coverslips in methanol for 2 h at -20°C. After washing with PBS, the cells on coverslips were stained by immersing in ActinGreen™ 488 RreadyProbes™ reagent (Invitrogen, Life Technologies) at room temperature for 90 min according to the manufacturer's suggestion. The traced dye was washed out from the cells by using PBS, and the coverslips were moved to glass slides and mounted with SlowFade™ Diamond Antifade Mountant (Invitrogen, Life Technologies). The images were then captured on a fluorescent microscope at ×40 magnification.
8
Immunofluorescence Imaging of TIBOV in Mosquito Midguts
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Midguts from infected mosquitoes were dissected (n = 30) at 14 dpi. The tissues were fixed using 4% paraformaldehyde for 1 h and washed with PBS containing 0.3% Triton X-100 (PBST) five times. Next, tissues were placed in blocking solution (PBS containing 5% goat serum and 0.3% Triton X-100) for 1 h and then incubated with primary rabbit anti-TIBOV-VP7 antibody (derived from rabbit serum, diluted 1:200 in PBST containing 5% goat serum) for 24 h, followed by secondary Cy3-conjugated goat anti-rabbit IgG (diluted 1:250 in PBST containing 5% goat serum; Abcam) for 12 h. The actin cytoskeleton was stained with Alexa Fluor 488 Phalloidin (Invitrogen) for 1 h. After each step, tissues were washed at least five times in 0.3% PBST to prevent the effects of reagents from affecting subsequent operations. Finally, tissues were mounted onto slides using SlowFade Diamond Antifade Mountant (Invitrogen), and images were recorded through a Leica SP8 confocal microscope (filter information TD 458/514/561, Leica, Germany). Using LAS X software (Leica), z-stack images were merged, and scale bars were added. PowerPoint 2019 was utilized for image grouping. All samples were analyzed under the same microscope and software settings.
9
Comprehensive Immunohistochemistry and RNA FISH Protocol
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IHC was performed with the following antibodies: Cleaved Caspase 3 (rabbit, R&D Systems, MAB835, 1:100), HOPX (mouse, Santa Cruz, sc-398703 1:00), GFP (chicken, Aves Labs, GFP-1020, 1:200 or goat, Abcam, ab6673, 1:200), Ki67 (mouse, BD Biosciences, 550609, 1:200), Lamp3 (DC-Lamp) (rat, Novus, DDX0191P-100, 1:100), NKX2.1 (Ttf1) (rabbit, Abcam, ab76013, 1:50), and SFTPC, (rabbit, Millipore Sigma, AB3786, 1:100). Slides were mounted using Slowfade Diamond Antifade Mountant (Invitrogen, catalog # S36972). RNA fluorescence in situ hybridization (FISH) was performed with RNAscope according the manufacturer’s instructions with the following probes: Mm-Col4a4-C2 (no. 1078041-C2), mm-Col4a3 (no. 544361), mm-Hopx-C2 (no. 405161-C2), mm-Igfbp7 (no. 425741), Mm-Lamb3 (no. 552161), and mm-Sftpc-C2 (no. 314101-C2). Fluorescent imaging was performed on a Zeiss LSM 710 confocal microscope with a 40 × water-immersion objective and processed using Fiji software (60 (link)). For cellular quantification of z-stacked images, at least 5 images or 200 cells in total were assessed per sample.
10
Lung Tissue Fixation and Staining Protocol
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