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Vectashield hardset mounting medium with dapi h 1500

Manufactured by Vector Laboratories
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Vectashield HardSet Mounting Medium with DAPI (H-1500) is a ready-to-use, permanent mounting medium for fluorescence microscopy. It contains the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) which specifically binds to DNA and emits blue fluorescence when excited. This product is designed to preserve fluorescent signals and harden over time to provide a durable mounting solution.

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10 protocols using vectashield hardset mounting medium with dapi h 1500

1

Fluorescent Dual-Label Immunohistochemistry

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For simultaneous visualization of both tracers, one series of brain tissue (n = 4) underwent fluorescent double-label immunohistochemical processing. Tissue was rinsed from the cryoprotectant storage solution with several washes in KPBS, and then incubated for 72 h at 4 °C in a blocking solution [KPBS containing 0.3 % Triton X-100, 2 % normal donkey serum (017-000-001; Jackson ImmunoRe-search, West Grove, PA, USA)], with both primary antibodies: anti-FG (1:10,000) and anti-CTB (1:5000). After rinses in KPBS, tissue was incubated for 1 h in the dark in the blocking solution containing the secondary antibodies: Alexa 488 anti-rabbit (1:200; A21206; Invitrogen, Carlsbad, CA, USA) and Alexa 546 anti-goat (1:200; A11056; Invitrogen, Carlsbad, CA, USA), both raised in donkey serum. Following rinses in KPBS, tissue was mounted in semidarkness onto slides (SuperFrost Plus), dried, coverslipped with Vectashield HardSet Mounting Medium with DAPI (H-1500; Vector Labs, Burlingame, CA, USA), and stored at 4 °C until analysis.
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2

Immunofluorescence Analysis of CD3 and Foxp3

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Sections of formalin-fixed paraffin-embedded hearts and frozen spleens were used for detection of CD3 and Foxp3 expression by immunofluorescence. First, paraffin-embedded sections were deparaffinized and submitted to a heat-induced antigen retrieval step by incubation in citrate buffer (pH 6.0). Then, sections were incubated overnight with the following primary antibodies: anti-CD3 (1:400; BD Biosciences) and anti-Foxp3 (1:400; Dako, Glostrup, Denmark). On the following day, secondary antibodies anti-goat IgG Alexa Fluor 488 (1:600; Molecular Probes) or anti-rabbit IgG Alexa Fluor 568 conjugated (1:100; Molecular Probes), diluted in 1% BSA in PBS, were added. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; VectaShield Hard Set mounting medium with DAPI H-1500; Vector Laboratories, Burlingame, CA). Images were analyzed using a confocal laser scanning microscope A1R (Nikon, Tokyo, Japan) and Image-Pro Plus version 7.01 (Media Cybernetics, Rockville, MD). Quantifications of CD3+/Foxp3+ cells percental area were performed in 10 fields randomly captured under x400 magnification, using Image-Pro Plus v.7.0.
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3

Macrophage Uptake of Oxidized LDL

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For immunohistochemistry, an anti-F4/80 antibody (Alexa Fluor® 647, Ab204467; Abcam, Cambridge, England) at a concentration of 1 : 250 was added to RPMI 1640 medium with 5% FBS, which contained 10 μg/mL Dil-ox-LDL (Highland Technology Center, MD, USA) with/without 10 nmol/L teneligliptin [11 (link)], and the incubation was continued for 18 hours at 37°C in 5% CO2. After washing, the stained macrophages were mounted in VECTASHIELD HardSet Mounting Medium with DAPI (H-1500, Vector Laboratories, CA, USA) and imaged with a BZ-X710 microscope/software (Keyence, Osaka, Japan).
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4

Immunohistochemical Staining of α-SMA

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The 5 µm frozen section was blocked with with 1% bovine serum albumin (A4503, Sigma-Aldrich) for 30 minutes. Then monoclonal anti-mouse α-SMA (A-2547, Sigma-Aldrich) was used at a dilution of 1∶5000 as primary antibody for 1 hour. The goat anti-mouse Rhodamine TRITC (M1061; Leinco Technologies) was used as secondary antibody with a dilution of 1∶500 for 1 hour at room temperature. Between each step, the slides were washed in PBS buffer 5 minutes for 3 times. The slides were rinsed then counterstained and mounted in Vectashield hard set mounting medium with DAPI (H-1500; Vector Laboratories).
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5

Immunohistochemical Staining of c-Fos

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Immediately following slicing, sections were incubated for 72 h at 4°C in a blocking solution [KPBS containing 0.3 % Triton X-100, 2 % normal donkey serum (017-000-001; Jackson ImmunoResearch, West Grove, PA, USA)], with the primary antibody: anti-c-Fos antibody raised in rabbit (1:10,000; Millipore, Billerica, MA). After rinses in KPBS, tissue was incubated for 1 h in the dark in the blocking solution containing the secondary antibody: anti-rabbit Alexa 488 (1:200; A21206; Invitrogen, Carlsbad, CA, USA). Following rinses in KPBS, in semidarkness tissue was mounted onto slides (SuperFrost Plus), dried, coverslipped with Vectashield HardSet Mounting Medium with DAPI (H-1500; Vector Labs, Burlingame, CA, USA), and stored at 4°C until analysis.
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6

Immunofluorescence Imaging of Chagasic Mouse Hearts

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The hearts obtained from chagasic mice were cryopreserved in Tissue-Tek (Sakura, Alphen aan den Rijn, The Netherlands), and 10-μm sections were obtained in Leica CM 1850 UV cryostat (Leica Microsystems, Wetzlar, Germany). To evaluate the presence of GFP+ cells in the heart, sections were incubated overnight with the following primary antibodies: anti-connexin 43 and troponin T (Santa Cruz Biotechnology, Inc.). On the following day, sections were incubated with Alexa fluor 594-conjugated anti-goat IgG or Alexa fluor 488-conjugated anti-rabbit IgG (Molecular Probes, Carlsbad, CA, USA). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) by using VectaShield Hard Set mounting medium with DAPI H-1500 (Vector Laboratories, Burlingame, CA, USA). Sections were then analyzed by using a FluoView 1000 confocal microscope (Olympus).
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7

Immunohistochemical Analysis of c-Fos Expression

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Immediately following slicing, sections were incubated for 72 h at 4 C in a blocking solution [KPBS containing 0.3% Triton X-100, 2% normal donkey serum (017-000-121; Jackson ImmunoResearch, West Grove, PA, USA)], with the primary antibody: c-Fos goat primary (1:2,000; sc-52-G; Santa Cruz, Dallas, TX). After rinses in KPBS, tissue was incubated for 1 h in the dark in the blocking solution containing the secondary antibody: Alexa 546 anti-goat (1:200; A11056; Invitrogen, Carlsbad, CA, USA). Following rinses in KPBS, in semidarkness tissue was mounted onto slides (SuperFrost Plus), dried, coverslipped with Vectashield HardSet Mounting Medium with DAPI (H-1500; Vector Labs, Burlingame, CA, USA), and stored at 4 C until analysis. This immunohistochemistry procedure was successfully repeated 8 times in the current study, and has been used successfully numerous additional times within our laboratory.
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8

Immunofluorescence Assay for Protein Localization

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Immunofluorescence assays were performed as described in [9 (link),53 (link)]. In brief, cells were seeded in chamber slides, washed with PBS, fixed with ice cold acetone, and permeabilized using 0.1% TritonX-100 (Sigma-Aldrich, St. Louis, MO, USA). After washing and blocking, the cells were incubated with a specific PDE6D antibody (1 in 200 dilution; Thermo Fisher Scientific) overnight. On the next day, the cells were incubated with a secondary antibody (Alexa-Fluor 488 anti-rabbit IgG; Thermo Fisher Scientific). Next, rinsing with PBS and mounting with Vectashield Hard Set Mounting Medium with DAPI H-1500 (Vector Laboratories, Burlingame, CA, USA) was performed.
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9

Immunohistochemical Analysis of Tissue Markers

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After deparaffinization, the sections were antigen-activated with Histo VT One (Nacalai Tesque) and blocked using Blocking One Histo (Nacalai Tesque). Primary antibody reactions were performed with anti-HPGDS (Cayman Chemicals, Ann Arbor, MI, USA) at a dilution ratio of 1:2000 in Solution A (Toyobo) and anti-Tryptase (ab2378: Abcam), anti-NG2 (sc-80003: Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-CD11b (66519-1-Ig: Proteintech Group, Inc., Rosemont, IL, USA), anti-Troponin T (MAB18742: Bio-Techne Corp., Minneapolis, MN, USA), anti-α-SMA (sc-53142: Santa Cruz Biotechnology, Inc.), and anti-CD31 (sc-376764: Santa Cruz Biotechnology, Inc.) at a dilution ratio of 1:500 in Solution A overnight at 4 °C. Secondary antibodies were reacted with goat anti-rabbit IgG H&L (Alexa Fluor® 647) (ab150083: Abcam) for anti-HPGDS and goat anti-mouse IgG H&L (Alexa Fluor® 488) (ab150113: Abcam) for others in Solution A (Toyobo) (dilution 1:1200) for 1 h at room temperature. Sections were mounted with Vectashield Hard Set Mounting Medium with DAPI (H-1500: Vector Laboratories, Inc., Newark, CA, USA). To confirm the specificity of the antibodies, we also verified that no fluorescence was observed in the negative control without the primary antibody.
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10

Immunofluorescent Detection of C3c in Ileum

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Tissues from the small intestine (ileum) were fixed overnight at 4°C in 4% paraformaldehyde, pH 7.2 and embedded in paraffin. Cross sections (5 microns) were de-paraffinized in xylene and rehydrated in serial dilutions of ethanol and 1xPBS. Tissues were blocked in 5% goat serum, 3% BSA, 0.3% Triton X100 in 1x PBS without antigen retrieval. Sections were incubated overnight at 4°C with a 1:100 dilution of a rabbit polyclonal anti-C3c antibody (ab15980, Abcam, Cambridge MA) in blocking solution. Sections were washed in 1xPBS and, for secondary detection, incubated with a 1:500 dilution of goat anti-rabbit AlexaFluor 555 (A-21429, Lifetechnologies, Grand Island NY) in blocking solution at room temperature for 1.5 hours. After washes in 1xPBS, coverslips were mounted and nuclei counterstained in Vectashield hardset mounting medium with DAPI (H-1500, Vector Laboratories, Burlingame CA). Images were taken with a Zeiss Axioplan2 and an AxioCam digital camera. Images were processed and layered with Photoshop CS5.
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