Vectashield hardset mounting medium with dapi

Manufactured by Vector Laboratories

Sourced in United States, Canada, Germany, United Kingdom

VECTASHIELD HardSet Mounting Medium with DAPI is a ready-to-use aqueous mounting medium that provides long-lasting performance for fluorescence microscopy applications. It is formulated to help preserve fluorescent signals and protect samples from photobleaching.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (6 mentions) Lsm 710 confocal microscope, by Zeiss (6 mentions) Cm3050, by Leica (4 mentions) 8 well chamber slide, by Merck Group (4 mentions) Lsm 700 microscope, by Zeiss (4 mentions) Paraformaldehyde (pfa), by Merck Group (4 mentions) Normal goat serum (ngs), by Vector Laboratories (4 mentions) Lsm 510, by Zeiss (4 mentions) Zen software, by Zeiss (3 mentions) Bz x810, by Keyence (3 mentions) Bio coated coverslips, by BD (3 mentions) Nf κb, by Santa Cruz Biotechnology (3 mentions) Hif 1α, by BD (3 mentions) Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (3 mentions) Dead end tunel kit, by Promega (3 mentions) Zen 2009 imaging software, by Zeiss (3 mentions) Lsm 880 inverted confocal microscope, by Zeiss (3 mentions) Poly l lysine (pll), by Merck Group (3 mentions) Dm6000 b widefield light microscope, by Leica (2 mentions) Fisherbrand superfrost, by Thermo Fisher Scientific (2 mentions)

139 protocols using vectashield hardset mounting medium with dapi

Visualizing DREADD Expression in Rat Brains

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Whole brains were extracted, fixed, cryoprotected, and sectioned into 40 μm coronal sections on a cryostat (Leica CM 3050 S, Leica Microsystems, Buffalo Grove, IL, USA). Sections were mounted on charged glass slides (FisherBrand Superfrost, ThermoFisher Scientific, Waltham, MA, USA) and coverslipped using HardSet VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) for microscopy. Fluorescence microscopy (Leica DM6000 B widefield light microscope, Leica Microsystems, Buffalo Grove, IL, USA) was used to verify mCherry expression (proxy for DREADD expression) in the hippocampal target cell bodies and terminal regions. The data of rats without bilateral mCherry expression in the subiculum were eliminated from analysis.

Lebonville C.L., Paniccia J.E., Parekh S.V., Wangler L.M., Jones M.E., Fuchs R.A, & Lysle D.T. (2020). EXPRESSION OF A HEROIN CONTEXTUALLY CONDITIONED IMMUNE EFFECT IN MALE RATS REQUIRES CAMKIIα-EXPRESSING NEURONS IN DORSAL, BUT NOT VENTRAL, SUBICULUM AND HIPPOCAMPAL CA1. Brain, behavior, and immunity, 89, 414-422.

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Tracing Neural Pathways via Microscopy

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At the end of the experiments, mice were anesthetized with an overdose of kethamine/xylazine (100/10 mg/kg) and transcardially perfused with phosphate-buffered saline (PBS) for 4 minutes followed immediately by 10% formalin. Brains were then collected and postfixed in 10% formalin for 24 h, followed by cryoprotection in 0.2 M Phosphate buffer (PB) at pH 7.4. Brains were sectioned using a vibratome into 40 μm slices and every other section was mounted on charged glass slides (FisherBrand Superfrost, Thermo Fisher Scientific, Waltham, MA, USA). After allowed to air dry under dark conditions, the mounted tissue was coverslip using HardSet VECTASHIELD mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Tissue that was not mounted was placed in cryopreserve solution and stored in a −20 °C freezer. The location of viral vector expression in the LC were evaluated through of the mCherry fluorescent tag and for cannulas placements in the RMTg were both verified using fluorescent microscopy (Leica DM6000 B widefield light microscope, Leica Microsystems, Buffalo Grove, IL, USA), as presented in Fig. 1. Only animals with bilateral mCherry expression in LC and correct bilateral cannula positions were included in statistical analyses.

Dornellas A.P., Burnham N.W., Luhn K.L., Petruzzi M.V., Thiele T.E, & Navarro M. (2021). Activation of Locus Coeruleus to Rostromedial Tegmental Nucleus (RMTg) Noradrenergic Pathway Blunts Binge-Like Ethanol Drinking and Induces Aversive Responses in Mice. Neuropharmacology, 199, 108797.

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TUNEL Assay for Apoptosis in Transplant Samples

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TUNEL was performed to determine the extent of apoptosis in brains transplanted with ISC-hpNSC. Sections were permeabilized with 0.1% Triton-X-100, in 0.1% sodium citrate for 5 min at 4 °C. The sections were washed twice with DPBS and then incubated with a labeling solution consisting of terminal deoxynucleotidyl transferase and nucleotide mixture in a ratio of 1:10 obtained from the In-Situ Cell Death Detection Kit-Fluorescein (Roche Applied Science, Indianapolis, IN) at 37 °C for 2 h. Sections were then washed three times with DPBS and mounted using VECTASHIELD Hard Set Mounting Medium with DAPI (Vector Laboratories).

Garitaonandia I., Gonzalez R., Christiansen-Weber T., Abramihina T., Poustovoitov M., Noskov A., Sherman G., Semechkin A., Snyder E, & Kern R. (2016). Neural Stem Cell Tumorigenicity and Biodistribution Assessment for Phase I Clinical Trial in Parkinson’s Disease. Scientific Reports, 6, 34478.

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Perfusion-fixation and Brain Sectioning

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Mice were anesthetized with an overdose of the anesthetic Tribromoethanol (Avertin, 1 mL, i.p.), and transcardially perfused with chilled 0.01 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were extracted and post-fixed in 4% PFA for 24 hours and then stored in PBS at 4°C. 45 μm coronal sections were collected using a Leica VT1000S vibratome (Leica Biosystems; Deer Park, IL, USA) and then stored in 0.02% sodium azide (Sigma Aldrich) in PBS. The tissue was then mounted on slides and allowed to dry before cover slipping with Vecta-Shield Hardset Mounting Medium with DAPI (Vector Laboratories; Newark, CA, USA). Viral placements were imaged at 4x magnification using a Keyence BZ-X800 All-in-one Fluorescence microscope (Keyence; Itasca, IL, USA).

C.A. G., G.B. G., H.L. H., D. P., T. S., S.L. D., K. B., W.P. K., C.W. H, & T.L. K. (2023). Disentangling the effects of Corticotrophin Releasing Factor and GABA release from the ventral bed nucleus of the stria terminalis on ethanol self-administration in mice. bioRxiv.

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Visualization of NHE and Cell Membrane

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Immunofluorescence stainings for visualization of inflammasomes were described in our previous publication^{31 (link)}. For visualization of NHE and cell membrane, BMDMs were stimulated with NHE component C_WT or C_mut. or C + B for 1 h, or C + B + A for 30 min, washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 1% BSA in PBS for 1 h. Cells were incubated with a rabbit sera anti-NHE-C (1:200 dilution in 1% BSA)^{55 (link)}, a mouse anti-NHE-B, or -A (1:200 dilution in 1% BSA)^{55 (link)}, an mouse anti-Histidine antibody (1:200 dilution in 1% BSA, ab18184, Abcam), and a rat fluorescein isothiocyanate-conjugated anti-CD11b antibody (1:200 dilution in 1% BSA, 101205, BioLegend) overnight at 4 °C. PBS containing 0.05% Tween-20 was used to wash between incubation steps. An anti-mouse secondary Rhodamine red antibody (115295146, Jackson ImmunoResearch) or rabbit secondary Rhodamine red antibody (111295144, Jackson ImmunoResearch) were used. The samples were mounted with VECTASHIELD Hardset Mounting Medium with DAPI (H-1500, Vector Laboratories, Inc.) and analyzed using a Leica SP5 confocal microscope.

Fox D., Mathur A., Xue Y., Liu Y., Tan W.H., Feng S., Pandey A., Ngo C., Hayward J.A., Atmosukarto I.I., Price J.D., Johnson M.D., Jessberger N., Robertson A.A., Burgio G., Tscharke D.C., Fox E.M., Leyton D.L., Kaakoush N.O., Märtlbauer E., Leppla S.H, & Man S.M. (2020). Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome. Nature Communications, 11, 760.

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Immunofluorescence Staining of AHR

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For immunofluorescence staining the Inside Stain Kit (Miltenyi Biotech Ltd., Bisley, Surrey, UK; Miltenyi Biotech Inc., Auburn, CA, USA) was used with minor modifications. After the fixation step, the cover glasses were incubated with the anti-AHR primary antibody (1:100, Santa Cruz Biotechnology) overnight at 4 °C. The slides were then washed and incubated with the secondary antibody for 30 min at room temperature (1:500, anti-rabbit FITC, Bethyl Laboratories, Inc., Montgomery, TX, USA). The slides were mounted in the VECTASHIELD HardSet Mounting Medium with DAPI (VECTOR LABORATORIES, INC., Burlingame, CA, USA), and then analysed using a confocal microscope (see below).

Tkachenko A., Henkler F., Brinkmann J., Sowada J., Genkinger D., Kern C., Tralau T, & Luch A. (2016). The Q-rich/PST domain of the AHR regulates both ligand-induced nuclear transport and nucleocytoplasmic shuttling. Scientific Reports, 6, 32009.

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Immunohistochemical Profiling of Rat Brain

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16 μM sections from frozen rat brains were fixed using 4% paraformaldehyde and rinsed twice in 1X PBS followed by permeabilization in 1X PBST for 30 mins. Slides were then transferred to a humidified chamber and blocked for 30 mins in 1XPBST/5% heat inactivated donkey serum, followed by incubation in the appropriate primary antibody: goat polyclonal anti-ZNF804A (S-16; Santa Cruz Biotechnology, Santa Cruz, CA) 1:50, rabbit polyclonal anti-GFAP antibody (abcam, Cambridge, MA) 1:1000, rabbit polyclonal anti-NeuN antibody (abcam) 1:500. Slides were incubated overnight at 4°C, washed 3 times in PBST, then blocked in 1XPBST/ 5% heat inactivated donkey serum. Sections were then incubated in secondary antibody and 1XPBST/ 5% heat inactivated donkey serum at 25°C for 2 hours. Secondary antibodies were Alexa Fluor 488 (1:50) and Alexa Fluor 546 (1:500; Molecular probes, Eugene, OR). Sections were then mounted in Vectashield HardSet mounting medium with DAPI (Vector laboratories, Burlingame, CA). Images were acquired on a Leica DMI 4000B inverted wide field fluorescence microscope at 20X. For negative controls, sections were incubated in non-immune serum followed by secondary antibody to check for non-specific binding.

Chang E.H., Kirtley A., Chandon T.S., Borger P., Husain-Krautter S., Vingtdeux V, & Malhotra A.K. (2015). Postnatal neurodevelopmental expression and glutamate-dependent regulation of the ZNF804A rodent homologue. Schizophrenia research, 168(0), 402-410.

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Lipid Droplet Quantification Assay

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Cells were cultured on eight wells chamber slides with or without 2% lipid mixture for 3 days and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing, cells were incubated with 3.8 µM of BODIPY 493/503 (Thermo Fisher Scientific, no. D3922) for 30 min at room temperature, then mounted with Vectashield hard-set mounting medium with DAPI (Vector laboratories, no. H-1500).

Okada N., Ueki C., Shimazaki M., Tsujimoto G., Kohno S., Muranaka H., Yoshikawa K, & Takahashi C. (2023). NFYA promotes malignant behavior of triple-negative breast cancer in mice through the regulation of lipid metabolism. Communications Biology, 6, 596.

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Xist RNA FISH in Differentiating ES Cells

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Xist RNA FISH was performed on ES cells, at day 5 of differentiation in our differentiation system, as previously described [6 (link)], with modifications. Xist RNA was detected with the 15 kb cDNA, pCMV-Xist-PA, as described [72 (link)]. The Xist probe was labelled with SpectrumGold dUTP (Vysis) by nick translation (Vysis). The cells were mounted in Vectashield HardSet mounting medium with DAPI (Vector Laboratories). Cells were visualised on the LSM 780 fitted with GaAsP detectors (Zeiss) using an α-Plan Apochromat 100X/1.46 oil objective (Zeiss). Images were analysed using the open source ImageJ distribution package, FIJI [73 (link)]. For each time point, 100–250 nuclei were analysed.

Keniry A., Gearing L.J., Jansz N., Liu J., Holik A.Z., Hickey P.F., Kinkel S.A., Moore D.L., Breslin K., Chen K., Liu R., Phillips C., Pakusch M., Biben C., Sheridan J.M., Kile B.T., Carmichael C., Ritchie M.E., Hilton D.J, & Blewitt M.E. (2016). Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing. Epigenetics & Chromatin, 9, 16.

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Visualizing β1-Integrin in 16HBE14o Cells

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16HBE14o cells were cultured on sterile coverslips, treated and fixed with 3.7% paraformaldehyde for 10 min at room temperature. Cells were incubated in blocking buffer (5% normal goat serum in 1 ×-PBS, 0.5% v/v Triton) for 1 h at room temperature. Cells were washed 3 times for 5 min in PBS between blocking, primary and secondary antibody incubations. Primary and secondary antibodies were diluted in blocking buffer (without Triton); anti-β1-integrin (JB1B) (Santa Cruz Biotech) 1:80 or Mouse IgG (goat anti-Mouse IgG – Texas Red) (Santa Cruz Biotech) 1:200. Phalloidin (Alexa 488) (Invitrogen) 1:1000 and Mouse IgG (Sigma) 1:80. Both primary and secondary antibody incubations were for 1 h at room temperature. Nuclear staining was carried out during mounting with VECTASHIELD Hard Set Mounting medium with DAPI (Vector Laboratories, Inc.). Images were obtained using an LSM710 Confocal Microscope (Zeiss Inc. UK).

Hennigan K., Conroy P.J., Walsh M.T., Amin M., O'Kennedy R., Ramasamy P., Gleich G.J., Siddiqui Z., Glynn S., McCabe O., Mooney C., Harvey B.J., Costello R.W, & McBryan J. (2016). Eosinophil peroxidase activates cells by HER2 receptor engagement and β1-integrin clustering with downstream MAPK cell signaling. Clinical Immunology (Orlando, Fla.), 171, 1-11.

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