Vectashield mounting medium with dapi

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Vectashield mounting medium with DAPI is a water-based fluorescent mounting medium designed to preserve fluorescent signals in immunostained or labeled samples. It contains the nuclear counterstain DAPI, which binds to DNA and emits blue fluorescence.

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955 protocols using vectashield mounting medium with dapi

Immunofluorescence of Rab35 and αSyn in Cell Cultures and Brain Organoids

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Cell cultures grown on coverslips were fixed with 4% PFA followed by permeabilization with 0.1% Triton X-100 and for 15 min at room temperature (RT) and then, blocked with 5% donkey serum for 1 h at RT. Primary antibody (rabbit anti-Rab35, 1:100; Abcam; rabbit anti-pser129 αSyn, 1:150; Abcam) diluted in antibody diluent (1% donkey serum + 0.1% Triton X-100) was incubated overnight at 4 °C. Secondary antibody (Goat-anti-rabbit Alexa 488, 1:500) was incubated for 1 h at RT. Cells were washed three times with PBS + 0.1% Tween-20. Coverslips were mounted on glass slides using VectaShield with DAPI mounting medium (Vector laboratories).
For whole brain organoids immunofluorescence, after being treated with 1 µM Alexa-633-tagged αSyn PFFs for 24 or 48 h, tissues were fixed in 4% PFA for 20 min at 4 °C followed by washing in PBS three times for 10 min before embedding in 4% low melting agarose (Sigma-Aldrich) in 1X PBS. Tissue sections of 50 µm were obtained using a vibratome (Leica VT1000 S Vibratome, Leica Microsystems, Wetzlar, Germany). For immunofluorescence, sections were blocked and permeabilized in 0.3% Triton X-100 and 4% FBS in PBS for 2 h at RT. Sections were then incubated with phalloidin Alexa-488 (Thermo Fisher, USA). Sections were washed three times with PBS + 0.1% Tween-20 and mounted on slides using VectaShield with DAPI mounting medium (Vector laboratories).

Rodrigues P.V., de Godoy J.V., Bosque B.P., Amorim Neto D.P., Tostes K., Palameta S., Garcia-Rosa S., Tonoli C.C., de Carvalho H.F, & de Castro Fonseca M. (2022). Transcellular propagation of fibrillar α-synuclein from enteroendocrine to neuronal cells requires cell-to-cell contact and is Rab35-dependent. Scientific Reports, 12, 4168.

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Meiotic Cell Fixation and Staining

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Fixed cells were prepared by treating 500–1000 μl of meiotic culture with 3.7% formaldehyde for 15 min at room temperature. Cells were permeabilized with either 1% Triton X-100 or 70% ethanol. (1) For Figure 4—figure supplements 1 and 2, cells were washed with 0.1 M potassium phosphate pH 6.4 and subsequently treated with 0.05 μg DAPI and 1% Triton in KPi sorbitol (0.1 M potassium phosphate, 1.2 M sorbitol, pH 7.5). Cells were then immediately washed with KPi sorbitol before imaging. (2) For Figure 6A–6B, cells were treated for five minutes with 1% Triton in KPi sorbitol and then resuspended in KPi sorbitol. Cells were then adhered on a poly-lysine treated multi-well slide and mounted with Vectashield Mounting Medium with DAPI (Vector Labs). (3) For Figures 5A–5B, 8C–8D and F, cells were washed with 0.1 M potassium phosphate pH 6.4 and then resuspended in KPi sorbitol buffer. Cells were then adhered to a poly-lysine treated multi-well slide, quickly permeabilized with 70% ethanol, and mounted with Vectashield Mounting Medium with DAPI (Vector Labs).

King G.A., Goodman J.S., Schick J.G., Chetlapalli K., Jorgens D.M., McDonald K.L, & Ünal E. (2019). Meiotic cellular rejuvenation is coupled to nuclear remodeling in budding yeast. eLife, 8, e47156.

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In Vivo Vascular Labeling and Brain Imaging

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24 h after the i.p. injection of Evans blue (1% dissolved in PBS; Nacarai Tesque), mice were transcardially perfused with PBS (under deep anesthesia) to remove the dye from the intraluminal space of blood vessels, and then with 4% paraformaldehyde in PBS (Nacarai Tesque). Brains were removed and post-fixed in 4% paraformaldehyde in PBS for 48 h. Sagittal sections of the brain (20 μm in thickness) were mounted on slides by using the Vectashield mounting medium with DAPI (Vector Laboratories Inc.). Mice were transcardially perfused with sulfo-NHS-biotin (0.5 mg/ml in PBS; Thermo Fisher Scientific) followed by 1% paraformaldehyde in PBS (Nacarai Tesque). Brains were removed and post-fixed in 4% paraformaldehyde in PBS for 48 h. The free-floating sagittal sections of the brain (20 μm in thickness) were washed in a PBS medium containing 0.05% Triton X-100 (PBS-T) and were then incubated with DyLight649-conjugated streptavidin (1:50; Vector Laboratories Inc.) in PBS-T, at 4°C, for 2 h. Sections on the slides were mounted by using the Vectashield mounting medium with DAPI (Vector Laboratories Inc.). Images were captured at room temperature with the use of a florescent microscope BIOREVO BZ-9000 system (Keyence) equipped with 10×/0.45-mm objective lens (CFI Plan Apochromat Lambda; Nikon Solutions Co. Ltd). BZ-II Viewer software was used for the acquisition of fluorescent images.

Yasuda T., Uchiyama T., Watanabe N., Ito N., Nakabayashi K., Mochizuki H, & Onodera M. (2023). Peripheral immune system modulates Purkinje cell degeneration in Niemann–Pick disease type C1. Life Science Alliance, 6(9), e202201881.

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Immunofluorescence Staining of Adherent Cells

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Cells with a inoculum density of 1 × 10⁵ cells/well were cultured onto coverslips (24-well plate, Fisher Scientific) pre-coated with collagen and fixed with 4% PFA after treatments as described above. The cells were washed with PBS 2 times and incubated with 0.3% Triton X-100 in PBS (PBS/Triton) at room temperature for 10 min. For β-actin staining, the treated cells were incubated with Phalloidin, CF488A (1:100 dilution in PBS, Biotium, Inc., Fremont, CA, USA) at room temperature for 1 h. The cells were then washed three times with PBS and mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). For ZO-1 staining, cells were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h and then incubated with an anti-rabbit ZO-1 polyclonal antibody (cat. # 61-7300, 1:100 dilution, Thermal Scientific, Ottawa, Canada) at 4 °C overnight. The cells were then washed three times with PBS and incubated with an Alexa fluor 488 goat anti-rabbit antibody (Thermal Scientific, cat. # A-11034) for 1 h at room temperature. Rinsed cells were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc.). The images were taken by a Zeiss Fluorescence Microscope (Carl Zeiss Ltd., Toronto, ON, Canada) [10 (link)].

Yang R., Hui Q., Jiang Q., Liu S., Zhang H., Wu J., Lin F., O K, & Yang C. (2019). Effect of Manitoba-Grown Red-Osier Dogwood Extracts on Recovering Caco-2 Cells from H2O2-Induced Oxidative Damage. Antioxidants, 8(8), 250.

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Immunofluorescence Staining of Cell Cultures

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Cells cultured on glass cover slips were fixed with 3% formaldehyde, permeabilized with 0.05% Triton-X and blocked with phosphate-buffered saline (PBS) containing 10% goat serum and 1% BSA. Cover slips were incubated overnight with primary antibody dilutions (1:250) prepared in 0.2% BSA in PBS 1 ×, and subsequently washed and incubated with an Alexa Fluor-488 goat anti-mouse antibody (Invitrogen, Waltham, MA, USA) for 1 h at room temperature. Slides were then exposed to DAPI (1 μg/ml) in PBS at room temperature for 5 min. Cover slips were mounted using VECTASHIELD with DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured utilizing a Leica Fluorescent microscope. IHC analysis was carried out as previously described;^{61 (link)} antibodies used were anti-E-cadherin, anti-fibronectin (GeneTex, Irvine, CA, USA) and anti-p27 (Cell Signaling, Danvers, MA, USA).

Dominguez C., Tsang K.Y, & Palena C. (2016). Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: rationale for combination therapies. Cell Death & Disease, 7(9), e2380-.

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ELOVL4 Subcellular Localization in HeLa Cells

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HeLa cells were grown on Labtek chamber slides and transiently transfected with ELOVL4 constructs. After 48 h, slides were rinsed and fixed as per Logan et al. (9 (link)). The slides were blocked with 5% nonfat dry milk and incubated with primary rabbit anti-HA antibody (Clonetech) and mouse anti-calnexin antibody (Abcam) overnight at 4°C. The following day, cells were washed and incubated with secondary anti-rabbit antibody conjugated with Alexa Fluor® 488 dye (Invitrogen) and anti-mouse antibody conjugated with Alexa Fluor® 568 dye (Invitrogen). The slides were then washed and coverslipped with Vectashield with DAPI mounting medium (Vector Labs) and imaged by confocal microscopy (Olympus FluoView 500, Olympus, Melville, NY).

Logan S., Agbaga M.P., Chan M.D., Brush R.S, & Anderson R.E. (2014). Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity. Journal of Lipid Research, 55(4), 698-708.

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Immunocytochemical Analysis of Neural Markers

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Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich). After 15–20 minutes, we washed cells with 0.1% BSA/PBS washing buffer 3 times and blocked for 1 hour by adding 0.3% triton X-100 (Sigma-Aldrich) and 10% normal goat serum (Invitrogen). After blocking, cells were incubated with primary antibodies at 4°C overnight. Anti-NG2 antibody (1: 200; Chemicon, Temecula, CA, USA), anti-Nestin antibody (1:500; BD Biosciences, NJ, USA), anti-Olig2 antibody (1:1000; Chemicon), anti-MBP antibody (1:1000; Abcam, Cambridge, UK), anti-A2B5 antibody (1:200; R&D systems), anti-PDGF-R antibody (1:200; R&D systems), and anti-O4 antibody (1:500; R&D systems) were used. Biotin was reacted for 30 minutes, and fluorescence-tagged antibody was reacted for one hour. Cells were mounted in VECTASHIELD with DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA).

Lee E.H, & Park C.H. (2017). Comparison of Reprogramming Methods for Generation of Induced-Oligodendrocyte Precursor Cells. Biomolecules & Therapeutics, 25(4), 362-366.

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Immunofluorescence Labeling of Tau and Aβ

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Immunofluorescence was performed on brain tissue from 8-M rTg4510 mice (FFPE) and MCI stage human tissue (free floats) (n = 3). All sections were subjected to antigen retrieval by 10mM sodium citrate pH 6.0 at 95°C for 10mins. Sections were washed in PBS containing 0.5% Triton X-100 and 10% goat serum/2%BSA/PBS-TritonX-100. Primary antibodies were diluted (see table below) and incubated overnight at 4°C. Subsequent to washing in PBS-Triton X-100, the sections were incubated in a mixture of Alexa-Fluor-546 goat anti-mouse IgM μ-chain specific (Invitrogen) 1:500 (for TOC1) and Alexa-Fluor-488 goat anti-mouse IgG (Invitrogen) 1:500 (for Ab39) secondary antibodies for 2h at room temperature. Alternatively, sections were counterstained with 1% Thioflavin-S as indicated. Sections were rinsed in PBS-Triton X-100. Free floating human brain sections from the entorhinal cortex were obtained from the Cognitive Neurology and Alzheimer’s Disease Center (CNADC) at Northwestern University and were mounted onto glass slides. Lipofuscin autofluorescence was eliminated by submerging the slides in Sudan Black (2 %). Free floats were then mounted using Vectashield with DAPI mounting medium (Vector) which also served to counterstain the nuclei. Staining was visualized using the Nikon C2 laser scanning confocal microscope.

Ward S.M., Himmelstein D.S., Ren Y., Fu Y., Yu X.W., Roberts K., Binder L.I, & Sahara N. (2014). TOC1: A valuable tool in assessing disease progression in the rTg4510 mouse model of tauopathy. Neurobiology of disease, 67, 37-48.

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Immunofluorescent Imaging of C2C12 Myotubes

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C2C12 myoblasts were seeded on 6-well plates with coverslips and differentiated into myotubes. Myotubes were rinsed in PBS, fixed in 4% paraformaldehyde for 15 min, and then washed three times with PBS and permeabilized in 0.1% Triton X-100 in PBS for 15 min. The myotubes blocked with 0.2% BSA for 30 min and then incubated with anti-MYH (sc-376157, Santa Cruz Biotechnology) overnight at 4 °C, followed by Alexa Fluor 488 (#A-21121, ThermoFisher) for 60 min. Coverslips were mounted with Vectashield with DAPI mounting medium (VECTOR Laboratories). Images were captured using a Nikon Eclipse Ti-U inverted microscope and Nikon DS-Ri2 camera using NIS-Elements software.

Lee S.M., Lee S.H., Jung Y., Lee Y., Yoon J.H., Choi J.Y., Hwang C.Y., Son Y.H., Park S.S., Hwang G.S., Lee K.P, & Kwon K.S. (2020). FABP3-mediated membrane lipid saturation alters fluidity and induces ER stress in skeletal muscle with aging. Nature Communications, 11, 5661.

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Immunofluorescence Analysis of Endothelial Cell Junctions

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HLMVEC (1 × 10⁵ cells) were seeded onto Lab‐Tek II chamber slides (Nalge Nunc International) and grown until 90% confluency was obtained as previously described.²⁵ Twenty‐four hours after treatment with cytomix with or without HMW or LMW HA, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. After additional washing, the cells were permeabilized by 0.1% Triton X‐100 and then blocked with 1% BSA at room temperature. For F‐actin staining, the slides were incubated with FITC‐phalloidin (Thermo Fischer Scientific) for 30 min at 37°C. For ZO‐1 staining, slides were incubated with Alexa ZO‐1 antibodies (5 μg/ml, Thermo Fischer Scientific) for 1 h at room temperature. For VE‐cadherin staining, primary antibodies to VE‐cadherin (1:100, Cell Signaling) were used. After multiple washing with PBS, slides were then incubated with the secondary antibody Alexa Fluor 488‐conjugated Goat Anti‐Rabbit IgG (H1L) DS Grade (1:50, Invitrogen, Thermo Fischer Scientific) for 1 h at room temperature. Slides were then mounted with Vectashield with DAPI mounting medium (Vector Laboratories). Images were obtained by AxioVision SE64 (Carl Zeiss Microscopy, LLC).

Sugita S., Naito Y., Zhou L., He H., Hao Q., Sakamoto A, & Lee J.W. (2022). Hyaluronic acid restored protein permeability across injured human lung microvascular endothelial cells. FASEB BioAdvances, 4(9), 619-631.

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