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Vectashield dapi mounting medium

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Vectashield-DAPI mounting medium is a water-based mounting medium designed for use with fluorescence microscopy. It is formulated to preserve fluorescence signals and reduce photobleaching. The medium contains 4',6-diamidino-2-phenylindole (DAPI), a fluorescent dye that binds to DNA and can be used to visualize cell nuclei.

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Lab products found in correlation

Lsm 700, by Zeiss (5 mentions) Hep 2 ana kit, by Inova Diagnostics (3 mentions) Paraformaldehyde (pfa), by Merck Group (3 mentions) Hemacolor rapid staining, by Merck Group (2 mentions) Lab tek chamber slide, by Thermo Fisher Scientific (2 mentions) Entellan resin, by Merck Group (2 mentions) Af 488 goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions) Zeissimager z1axiocammrm, by Zeiss (2 mentions) Antibody diluent, by Agilent Technologies (2 mentions) Rat anti mouse cd45r, by BD (2 mentions) Alexa fluor 488 donkey anti goat igg h l, by Thermo Fisher Scientific (2 mentions) Goat polyclonal igg anti mouse cd3ε, by Santa Cruz Biotechnology (2 mentions) Alexa fluor 594 donkey anti rat igg h l, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (2 mentions) Cfi planapo lamda objective, by Nikon (2 mentions) Eclipse ni e microscope, by Nikon (2 mentions) Axiocam mrm, by Zeiss (2 mentions) Bodipy, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

Vectashield mounting medium containing DAPI VECTASHIELD Mounting Medium with 4’,6-diamino-2-phenylindole (DAPI) VECTASHIELD Antifade Mounting Medium with DAPI VECTASHIELD® Mounting Medium with 40,6-diamidino-2-phenylindole (DAPI; VECTASHIELD HardSet Mounting Medium with DAPI VECTASHIELD antifade mounting medium with 4′,6‐diamidino‐2‐phenylindole (DAPI, H‐1200 Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) Vectashield HardSet Mounting Medium with DAPI (H-1500; Vectashield mounting medium including DAPI Vectashield mounting medium containing DAPI for nuclear staining

55 protocols using vectashield dapi mounting medium

1

Histological Analysis of Lymph Nodes

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For histological analysis, fixed lymph nodes were dehydrated in 100% ethanol and embedded in paraffin (Leica) using a Leica EG1150 tissue embedder. 7-μm-thick sections using a HM340E Thermo Scientific microtome were processed for hematoxylin-eosin (HE) (Leica) and immunohistochemistry staining as described (41 (link)). For immunohistochemistry of histology slides the following antibodies were used: anti-TER-119 (R&D Systems, MAB1125) and Alexa Fluor 488 conjugated goat anti-rat IgG antibody (Life Technologies A11006). Stained slides were mounted with Vectashield DAPI Mounting Medium (Vector Laboratories, H-1200). Tissue section samples were imaged using a Nikon Ni-U upright microscope (Nikon Instruments) using a 40x dry objective connected to a Nikon DS-Ri2 camera.
Bálint L., Ocskay Z., Deák B.A., Aradi P, & Jakus Z. (2020). Lymph Flow Induces the Postnatal Formation of Mature and Functional Meningeal Lymphatic Vessels. Frontiers in Immunology, 10, 3043.
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2

Spermatogonia C-kit Expression Analysis

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Spermatogonia were fixed by using 4% paraformaldehyde, permeabilized, and incubated with 5% goat serum dissolved in 0.2% Triton X-100 PBS buffer was used for blocking. Then, the samples were incubated with the primary antibody against the C-kit (1 : 200, Bioss, Beijing, China) and the fluorescently labelled secondary antibody (Bioss, Beijing, China), with nuclei counterstained with VECTASHIELD DAPI mounting medium (Vector Labs, California, USA). Last, images were obtained using confocal microscopy (MSHOT, Guangzhou, China).
Tang S., Yang P., Ding Y., Chen Q., Huang H., Chen X, & Zhou H. (2022). Silencing CAMK2D Promotes the Proliferation of Spermatogonia in the Testis of Experimental Varicocele Rats. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 7121245.
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3

Quantifying Skin Inflammation and Wound Healing

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Immunohistochemistry was performed on formalin-fixed paraffin-embedded mouse skin samples using the avidin-biotin complex method and AEC development (Vector Laboratories). Indicated antibodies were applied overnight. Sections were counterstained with hematoxylin. Images were captured at 40X magnification using a Nikon Optiphot microscope and Nikon Elements F software. Histology was assessed by H&E after IL-6 addition. The epidermal thickness from the basal layer keratinocytes to beginning of stratum corneum in three locations per healed mouse wound in multiple histology sections was measured by Image J software.
Immunocytochemistry was performed on NHEKs plated on plated on collagen-coated coverslips and treated with 20 μg/ml poly (I:C) as above. Fixed cells were incubated with primary antibodies overnight, appropriate Alexa Fluor secondary antibodies and counterstained using VectaShield DAPI mounting medium (Vector Labs, Burlingame, CA). Slides were imaged at 60x magnification using the Nikon C1si True Spectral Imaging Confocal Laser Scanning Microscope system (Cell Imaging Core Facility, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins, Baltimore, MD). Cell morphology and beta-catenin nuclear localization were quantified using the CellProfiler image analysis software (www.cellprofiler.org) (Carpenter et al., 2006 (link)) from confocal images of nuclei.
Nelson A.M., Reddy S.K., Ratliff T.S., Hossain M.Z., Katseff A.S., Zhu A.S., Chang E., Resnik S.R., Page C., Kim D., Whittam A.J., Miller L.S, & Garza L.A. (2015). dsRNA Released by Tissue Damage Activates TLR3 to Drive Skin Regeneration. Cell stem cell, 17(2), 139-151.
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4

Cell Morphology Evaluation Protocol

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Cells (3 × 104/per well) were seeded in Lab-Tek™ chamber slides (Thermo-Scientific®, Waltham, MA, USA) with 400 µL of supplemented medium (Sigma®, St. Louis, MO, USA) and incubated for 24 h. The treatment duration with IC50 of samples/controls was 24 h. Then, cell fixation began with the removal of the culture medium and the addition of 2% paraformaldehyde (Sigma®, St. Louis, MO, USA) for 30 min at 37 °C. Morphology changes were evaluated by: i) Hemacolor® rapid staining (Merck®, Darmstadt, D.E.) or ii) F-actin staining with Rhodamine-Phalloidin Reagent (Thermo-Fisher™); according to each manufacturer’s instructions. Later, slides were prepared with Entellan® resin (Merck®, Darmstadt, D.E.) or Vectashield®/DAPI mounting medium (Vector Laboratories®), depending on each assay. Finally, observations were conducted using light microscopy (BX41, Olympus®, Miami, FL, USA) or confocal microscopy (LSM 700, Zeiss®, Pleasanton, CA, USA) [30 (link)].
Varela-Rodríguez L., Sánchez-Ramírez B., Saenz-Pardo-Reyes E., Ordaz-Ortiz J.J., Castellanos-Mijangos R.D., Hernández-Ramírez V.I., Cerda-García-Rojas C.M., González-Horta C, & Talamás-Rohana P. (2021). Antineoplastic Activity of Rhus trilobata Nutt. (Anacardiaceae) against Ovarian Cancer and Identification of Active Metabolites in This Pathology. Plants, 10(10), 2074.
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5

Transient Transfection of EGFP Constructs in HeLa Cells

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Transient transfections were performed in HeLa cells (ATCC) as previously described (Onder and Moroianu, 2014 (link)). HeLa cells plated at 50-70% confluency on 12mm poly-L-lysine coated glass coverslips 24 hours before transfection were transiently transfected with the EGFP construct of interest (as indicated in the figure legends) using FuGENE 6 Transfection Reagent (Roche Applied Science, IN). The media was changed to DMEM with 10% FBS and 1% penicillin-streptomycin after 6 hours and the cells were fixed with 3.7% formaldehyde in PBS for 10 minutes. Coverslips were mounted with Vectashield-DAPI mounting medium (Vector Labs, CA) to identify the nuclei of cells. Fixed cells were analyzed with a Leica TCS Sp5 broadband confocal microscope using a 63X immersion oil objective and 1.6× optical zoom and representative images were taken using Leica LAS AF software (Leica Microsystems). The intracellular localization of EGFP-tagged proteins was phenotyped as either mostly nuclear, pancellular (both in the cytoplasm and nucleus with no accumulation in either compartments), or mostly cytoplasmic and data from at least 4 experiments were quantitated and graphical representations were used to display average values and standard deviations.
Onder Z., Chang V, & Moroianu J. (2014). Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway. Virology, 474, 28-33.
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6

Apoptosis Assay for Adherent Cells

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Adherent cultures plated at 3 × 104 cells per well in Lab-Tek™ chamber slides (Thermo Scientific®) were treated with the IC50 of samples and controls (25 μg/mL vincristine Sigma® and 1X PBS vehicle) for 24 h. Then, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde (Sigma®) for 1 h at 37 °C. The cells were then washed with 1X PBS and stained. The morphology of the cells was determined using Hemacolor® rapid staining (Merck®), and the presence of apoptosis was assessed using the Apo-BrdU™ TUNEL assay kit with Alexa Fluor 488 (Invitrogen®), according to the manufacturer’s instructions. After applying both stains, the slides were treated with Vectashield®/DAPI mounting medium (Vector Laboratories®) or Entellan® resin (Merck®) according to the assay and analyzed by optical microscopy (BX41, Olympus®) or confocal microscopy (LSM 700, Zeiss®) using a 40X immersion lens with Image-Pro Plus (version 4.0, Media Cybernetics©) or ZEN 2011 (version 1.0, Zeiss®) software.
Varela-Rodríguez L., Sánchez-Ramírez B., Rodríguez-Reyna I.S., Ordaz-Ortiz J.J., Chávez-Flores D., Salas-Muñoz E., Osorio-Trujillo J.C., Ramos-Martínez E, & Talamás-Rohana P. (2019). Biological and toxicological evaluation of Rhus trilobata Nutt. (Anacardiaceae) used traditionally in mexico against cancer. BMC Complementary and Alternative Medicine, 19, 153.
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7

Immunofluorescence Staining of Cleaved Caspase 3

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Immunofluorescence was performed as recently described29 (link). Cells were fixed with 3% formaldehyde/PBS for 30 min, permeabilized with 0.5% Triton X-100/PBS for 10 min and blocked with 3% BSA/PBS for 1 h. Staining of cleaved caspase 3 (Cell Signaling, #9664) was carried out overnight at 4 °C and with anti-rabbit secondary antibody for 2 h at room temperature. After several washes with PBS, samples were covered with Vectashield/DAPI mounting medium (Vector Labs). Images were acquired using an AxioImager.Z1/ApoTome microscope (Zeiss).
Eke I., Aryankalayil M.J., Bylicky M.A., Makinde A.Y., Liotta L., Calvert V., Petricoin E.F., Graves E.E, & Coleman C.N. (2022). Radiotherapy alters expression of molecular targets in prostate cancer in a fractionation- and time-dependent manner. Scientific Reports, 12, 3500.
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8

Detection of Anti-nuclear Antibodies in Mice

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Anti-nuclear antibodies were detected in the sera of mice by using the HEp-2 ANA kit (Inova Diagnostics, Inc., San Diego, CA, USA). All procedures were performed according to the manufacturer’s instructions. Mouse sera were diluted at 1:80 and incubated on HEp-2-fixed substrate slides for 1 h at room temperature in a humidified chamber. After three 5-min washes with PBS, the substrate slides were treated with a 1:100 dilution of AF 488 goat anti-mouse IgG (H + L) (Life Technologies) for 45 min at room temperature. After three washes, slides were treated with Vectashield DAPI-mounting medium (Vector Laboratories) and overlaid with glass coverslips. Fluorescence was detected by fluorescence microscopy at 20× magnification using a Nikon microscope, and all images were obtained with exposure of 200 milliseconds.
Gupta S., Li D., Ostrov D.A, & Nguyen C.Q. (2021). Blocking I—Ag7 class II major histocompatibility complex by drug-like small molecules alleviated Sjögren’s syndrome in NOD mice. Life sciences, 288, 120182.
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9

BrdU Incorporation Assay Protocol

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The E, M and RE cells were incubated with 1 μL of BrdU solution (ThermoFisher, Waltham, MA, USA) for 90 min, washed with a phosphate-buffered saline solution (PBS) and fixed using 4% formaldehyde (Sigma-Aldrich, Burlington, VT, USA). Next, the cells were treated with 2M-HCl (Merck, Burlington, VT, USA), washed with PBS-0, 5% Tween 20–0 and 5% BSA (Sigma-Aldrich, Burlington, VT, USA) and incubated with anti-BrdU antibody (1:10, ThermoFisher, Waltham, MA, USA) and anti-mouse Ig-FITC (1:100, ThermoFisher, Waltham, MA, USA). The coverslips were mounted using Vectashield-DAPI mounting medium (Vector Laboratories, Burlingam, CA, USA). Images were taken with ZeissImager.Z1AxioCamMRm (Zeiss, Oberkochen, Germany), and the stained nuclei were counted. Statistical analysis was performed using the Mann–Whitney test [28 (link)].
Santos M., Ferreira M., Oliveira P., Mendes N., André A., Vieira A.F., Nunes J.B., Carvalho J., Rocha S., Azevedo M., Ferreira D., Reis I., Vinagre J., Paredes J., Heravi-Moussavi A., Lima J., Máximo V., Burleigh A., Roskelley C., Carneiro F., Huntsman D, & Oliveira C. (2022). Epithelial-Mesenchymal Plasticity Induced by Discontinuous Exposure to TGFβ1 Promotes Tumour Growth. Biology, 11(7), 1046.
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10

Investigating HAMLET and TLR Interactions

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0.1×106 RAW264.7 cells or primary human monocytes were seeded per well in chamber slides and differentiated to Mo‐DCs and Mo‐M. In one set of experiments, the cells were treated or not with 10 μg/ml PmB for 2‐h and stimulated with HAMLET, αLA or LPS, as described above and incubated a further 18‐h. In a second set of experiments, the cells were treated with 0.2 mg/ml of Alexa Fluor 568‐conjugated HAMLET or αLA (labeled using the Alexa Fluor 568 labeling kit from ThermoFisher according to the manufacturer's instructions) for 2‐h and washed. For both sets of experiments, the cells were then fixed in 4% paraformaldehyde (PFA), permeabilized in 0.1% Triton‐X100 in PBS and stained with TLR4 (clone 25) 1:100 or TLR2 (clone A‐9) 1:100 (both from Santa Cruz Biotechnology) overnight at 4°C after which the cells were incubated with goat‐anti‐mouse Alexa Fluor 488‐conjugated secondary antibody 1:200 for 1‐h. Vectashield DAPI mounting medium (Vector Laboratories, Inc.) was used for nuclear staining. The cells were visualized using an Olympus BX41 microscope and images were collected by the cellSense Dimension v1.9 software using an Olympus DP72 CCD camera (Olympus).
Vansarla G., Håkansson A.P, & Bergenfelz C. (2021). HAMLET a human milk protein‐lipid complex induces a pro‐inflammatory phenotype of myeloid cells. European Journal of Immunology, 51(4), 965-977.
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