Vectashield fluorescence mounting medium

Manufactured by Vector Laboratories

Sourced in United States

Vectashield is a high-performance fluorescence mounting medium designed for use with a wide range of fluorescent-labeled samples. It is optimized to preserve and enhance fluorescence signals, making it suitable for various microscopy applications.

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Lab products found in correlation

Apotome, by Zeiss (4 mentions) Axiocam, by Zeiss (2 mentions) Axiovision, by Zeiss (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Goat anti c myc, by Novus Biologicals (1 mentions) Chicken anti green fluorescent protein gfp, by Aves Labs (1 mentions) Inform software, by PerkinElmer (1 mentions) Vectra multispectral imaging system, by PerkinElmer (1 mentions) Spectral dapi, by PerkinElmer (1 mentions) Mrc 1024, by Bio-Rad (1 mentions) Eclipse e600 microscope, by Nikon (1 mentions) Propidium iodide, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Ez retriever, by BioGenex (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Lamp 1 antibody, by Abcam (1 mentions) Zen microscope software, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Alexa 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)

13 protocols using vectashield fluorescence mounting medium

Immunolabeling of H2A.X and D2 Receptor

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The protocols used were identical to those described above for each tissue type, up to and including the primary antibody incubation stage. Two secondary antibodies were applied either the IRDye^™ 800CW conjugated donkey‐anti‐rabbit IgG (H+L, 1 : 1000) or IRDye^™ 680RD conjugated goat‐anti‐rabbit IgG (H+L, 1 : 1000), with the former used for labelling of the H2A.X protein and both IR dyes used for labelling the D2 receptor. Sections were incubated for 1 h in the dark, and thoroughly washed in PBS–BSA buffer prior to being coverslipped using Vectashield fluorescence mounting medium (Vector Laboratories, Peterborough, UK).

Eaton S.L., Cumyn E., King D., Kline R.A., Carpanini S.M., Del‐Pozo J., Barron R, & Wishart T.M. (2015). Quantitative imaging of tissue sections using infrared scanning technology. Journal of Anatomy, 228(1), 203-213.

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Cornea Visualization and Immunohistochemistry

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Reporter visualization. Mice were sacrificed by CO2 asphyxiation, and their eyes were isolated and typically fixed in 2% paraformaldehyde in sodium acetate buffer, pH 6.0 [12 (link)]. The corneas surgically dissected from the P0-Cre;R26-tdTomato animals were fixed in 4% paraformaldehyde and examined directly following mounting, without antibody-mediated enhancement. In the case of the MADM mice, whole corneas were incubated overnight at 4 °C in combined primary antibodies (chicken anti-green fluorescent protein [GFP]; Aves Labs, Tigard, OR; 1:500, and goat anti-c-Myc; Novus Biologicals, Littleton, CO; 1:200) diluted in Tris-buffered saline (TBS), pH 7.3, with 1.0% bovine serum albumin (BSA) and 0.4% Triton X-100. Anti-c-Myc antibody was preabsorbed with fixed wild-type tissue before use [13 (link)]. The following day, the slides were labeled serially with fluorescein isothiocyanate (FITC)–conjugated and Alexa Fluor 555–conjugated secondary antibodies (1:200 and 1:400 dilution, respectively) to enhance visualization of GFP and c-Myc-RFP, respectively. After several buffer rinses, four radial incisions were made in each cornea to produce “petals,” and the resulting flatmounts were placed on glass slides with the endothelium facing up and then coverslipped using Vectashield fluorescence mounting medium (Vector Labs, Burlingame, CA) [14 (link)].

Harrison T.A., He Z., Boggs K., Thuret G., Liu H.X, & Defoe D.M. (2016). Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area. Molecular Vision, 22, 31-39.

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Multiplex Immunofluorescent Staining of TMAs

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Immunofluorescent staining was performed using the opal multiplex immuno-fluorescent system (Perkin Elmer, Hopkinton, MA). Briefly, TMA slides were deparaffinized and tissues were fixed with 10% neutral buffered formalin. Heat- induced epitope retrieval (HIER) was performed in a conventional microwave oven using citrate buffer (pH 6.0). Each section was subjected to six sequential rounds of staining (pan-cytokeratin, DAPI, CD8, CD4, Foxp3, and CD68). Each round of staining included a protein block followed by staining with primary antibody and its corresponding secondary HRP- conjugated polymer. Signal amplification was achieved by using a specific TSA (Tyramide Signal amplification) solution for each antibody. Following this covalent reaction between the labeled tyramide and the tissue, the TMA slides were further subjected to HIER to remove unbound antibodies before the slides were stained with the next antibody. After all five sequential antibody staining, slides were counterstained with spectral DAPI (Perkin Elmer, Hopkinton, MA) and mounted with vectashield fluorescence mounting medium (Vector Labs, Burlingame, CA). Multiplex stained TMA slides were imaged using Vectra multispectral imaging system (Perkin Elmer, Hopkinton, MA) and the images were analyzed using inform software (Perkin Elmer, Hopkinton, MA)

Barua S., Fang P., Sharma A., Fujimoto J., Wistuba I., Rao A.U, & Lin S.H. (2018). Spatial Interaction of Tumor Cells and Regulatory T cells Correlates with Survival in Non-Small Cell Lung Cancer. Lung cancer (Amsterdam, Netherlands), 117, 73-79.

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Immunofluorescence Staining Protocol

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Cells were seeded on glass coverslips and fixed with either 4% w/v paraformaldehyde for 10 min or ice-cold methanol for 10 min. Fixed cells were further permeabilized with 0.1% v/v Triton X-100 for 10 min when needed. Nonspecific binding was blocked with 5% w/v bovine serum albumin (BSA, Merck KGaA, Darmstadt, Germany) and 5% v/v nonimmune serum from the host species of secondary antibody. Primary antibodies were applied overnight in 1% w/v BSA at 4 °C (Table S2). After washing in PBS, coverslips were incubated with fluorescent-labeled secondary antibodies in 1% w/v BSA for 1 h at room temperature (Table S2). Cells were washed and mounted in Vectashield fluorescence mounting medium containing DAPI (Vector Laboratories Inc., Burlingame, CA, USA) or propidium iodide (Merck KGaA). Immunofluorescence images were either captured on a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan) connected to the Lucia Cytogenetics v1.5.6 software (Laboratory Imaging, Prague, Czech Republic), or with a Bio-Rad MRC 1024 (Bio-Rad Laboratories Inc.) confocal laser microscope.

Hollósi P., Váncza L., Karászi K., Dobos K., Péterfia B., Tátrai E., Tátrai P., Szarvas T., Paku S., Szilák L, & Kovalszky I. (2020). Syndecan-1 Promotes Hepatocyte-Like Differentiation of Hepatoma Cells Targeting Ets-1 and AP-1. Biomolecules, 10(10), 1356.

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Multiplex Immunofluorescence Staining

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In all, 5 μm sections obtained from the TMA blocks were deparaffinized and tissues were fixed with formaldehyde:methanol (1:10) prior to antigen retrieval in heated Citric Acid Buffer (pH 6.0) for 15 min (EZ Retriever microwave, BioGenex). Each section was put through seven sequential rounds of staining, each including a protein block with 1% BSA followed by primary antibody and corresponding secondary horseradish peroxidase-conjugated polymer (Table 3 and Supplementary Fig. 1A). Each horseradish peroxidase-conjugated polymer mediated the covalent binding of a different fluorophore using tyramide signal amplification (Table 3 and Supplementary Fig. 1A). This covalent reaction was followed by additional antigen retrieval in heated Citric Acid Buffer (pH 6.0) for 15 min to remove bound antibodies before the next step in the sequence. After all seven sequential reactions, sections were counterstained with DAPI (Life Tech) and mounted with Vectashield fluorescence mounting medium (Vector Labs, Burlingame, CA).

Carstens J.L., Correa de Sampaio P., Yang D., Barua S., Wang H., Rao A., Allison J.P., LeBleu V.S, & Kalluri R. (2017). Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nature Communications, 8, 15095.

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Quantifying CuNCs-FC131-TR Internalization in 4T1 Cells

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To determine the internalization of CuNCs-FC131-TR in 4T1 cells, immunofluorescent staining was performed. After the incubation of CuNCs-FC131-TR in 4T1 cells for indicated times, cells were washed twice in PBS, then fixed for 15 min in 4% PFA. After washing three times, cells were blocked/permeabilized in Roth Buffer for 1 h (0.25% Milk, 1% BSA, 0.3% Triton-X 100 in TBS (50 mM Tris/150 mM NaCl; pH 7.4). Lamp1 antibody (Abcam, ab24170) was diluted in Roth buffer (1:250) and incubated with cells at room temperature for 2 h. After washing, cells were incubated with Alexa Flour 488 Conjugated Donkey anti Rabbit IgG (Life Tech, A-21206) (diluted in Roth buffer, 1:250) for 1 h. With another washing and DAPI (Sigma D9542) staining, the cell-growing coverslip was put on the VECTASHIELD fluorescence mounting medium (Vector Labs, H-1000) and then fixed with nail polish. A Zeiss LSM-700 confocal microscope was used for imaging; regions of interest (ROIs) and thresholds were determined, and signal over the threshold was quantified using ZEN microscope software (Carl Zeiss Microscopy).

Heo G.S., Zhao Y., Sultan D., Zhang X., Detering L., Luehmann H.P., Zhang X., Li R., Choksi A., Sharp S., Levingston S., Reichert D.E., Sun G., Razani B., Li S., Weilbaecher K.N., Dehdashti F., Wooley K.L, & Liu Y. (2019). Assessment of Copper Nanoclusters for Accurate In Vivo Tumor Imaging and Potential for Translation. ACS applied materials & interfaces, 11(22), 19669-19678.

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Immunofluorescence Staining of α-Tubulin

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Immunofluorescence was performed as described [28 (link)]. The primary antibody was anti-α-Tubulin (Cell Signaling; mouse monoclonal; 1:2000) and the secondary antibody was Alexa488-conjugated anti-mouse IgG (Molecular Probes; 1:500). The slides were mounted in VectaShield fluorescence mounting medium containing 4, 6-diamidino-2-phenylindole (Vector Laboratories). Images were analyzed using a Zeiss confocal laser scanning microscope. Alternatively, cells were stained with α-tubulin antibody (1:50, mouse monoclonal; Abcam) followed by a CY5-conjugated secondary anti-mouse antibody (1:200; Abcam). The cover slips were mounted with Invitrogen Anti Fade Mounting Medium containing DAPI and imaged using a Zeiss AxioImager.Z1 microscope at 63x magnification.

Rajasekaran D., Siddiq A., Willoughby J.L., Biagi J.M., Christadore L.M., Yunes S.A., Gredler R., Jariwala N., Robertson C.L., Akiel M.A., Shen X.N., Subler M.A., Windle J.J., Schaus S.E., Fisher P.B., Hansen U, & Sarkar D. (2015). Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): Evaluation using an endogenous HCC model. Oncotarget, 6(28), 26266-26277.

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Quantifying Mitotic Cells via Histone H3

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Cells were seeded on coverslips and fixed with 4% paraformaldehyde in PBS, permeabilized with PBS containing 0.1% Triton-X-100, and blocked in PBS containing 0.1% bovine serum albumin (BSA) and 5% normal donkey serum for 2 h at room temperature. Coverslips were incubated with rabbit anti-phosphorylated histone H3 (Ser10) (Upstate Biotechnology; Lake Placid, NY, USA) diluted 1:500 in PBS containing 0.1% BSA overnight at room temperature. Following washes with PBS, coverslips were incubated with Alexa-Fluor Donkey 555 anti-Rabbit (Life Technologies; Carlsbad, CA, USA) diluted 1:500 in PBS 0.1% BSA for 1 h at room temperature protected from light. Coverslips were then washed, stained with DAPI, and mounted on slides with Vectashield fluorescence mounting medium (Vector Laboratories; Burlingame, CA, USA). Cells were visualized using an Olympus BX61 fluorescence microscope coupled with a Hamamatsu ORCA-ER camera and using SlideBook software (Intelligent Innovations, Inc., Denver, CO, USA). The percent of cells positive for phosphorylated histone H3 was quantified.

Crowe E.P., Tuzer F., Gregory B.D., Donahue G., Gosai S.J., Cohen J., Leung Y.Y., Yetkin E., Nativio R., Wang L.S., Sell C., Bonini N.M., Berger S.L., Johnson F.B, & Torres C. (2016). Changes in the Transcriptome of Human Astrocytes Accompanying Oxidative Stress-Induced Senescence. Frontiers in Aging Neuroscience, 8, 208.

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Visualizing Retinal Pigment Epithelium Flat-Mounts

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In order to simultaneously view all the cells within the RPE layer, epithelial flat-mounts were prepared. Briefly, enucleated eyes were fixed in 4% PFA for overnight at 4°C. After several rinses in PBS, eyecups were prepared by making a circumferential incision at posterior to the ora serrata, and the anterior segments, i.e., cornea, ciliary body, and lens, were discarded. The neural retina was subsequently peeled away from these eyecups, resulting in an intact epithelium partitioning with the choroid, to which it is adherent. The resulting RPE-choroid-sclera whole-mounts were labeled using primary antibodies followed by fluorescent-dye conjugated secondary antibodies. After several tris-buffered saline (TBS) rinses, flat-mounts were placed on glass slides with the epithelium facing up and mounted in Vectashield fluorescence mounting medium (Vector Labs, Burlingame, CA, USA),and examined using a Zeiss Axio Imayger M2 system equipped with ApoTome, AxioCam and AxioVision (Zeiss, Peabody, MA, USA). In certain cases, nuclear staining was additionally carried out by exposure to propidium iodide (0.1 mg/ml in TBS, containing 0.5 mg/ml RNAase A; Roche, Basel, Switzerland). To ensure that the retina laid flat, several radial cuts (5–6, depending on the size of the eye) were made in the tissue from the periphery to the optic nerve area,

Lu Q., Scott P.A., Vukmanic E.V., Kaplan H.J., Dean D.C, & Li Q. (2020). Yap1 is required for maintenance of adult RPE differentiation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(5), 6757-6768.

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Immunolabeling of Kisspeptin and tdTomato in Mouse Brain

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Immunofluorescence labelling was performed on free‐floating coronal brain sections. For kisspeptin and tdTomato immunostaining, sections were incubated overnight with a sheep anti‐kisspeptin‐10 antiserum (AC024, dilution 1 : 2000; gift from Alain Caraty) combined with a rabbit anti‐RFP antibody (dilution 1 : 2000; Rockland Immunochemicals, Pottstown, PA, USA) in TBS containing 2% normal donkey serum, 0.3% Triton‐X‐100 and 0.25% bovine serum albumin. After several washes with TBS, the sections were placed in biotinylated donkey anti‐sheep secondary immunoglobulins (dilution 1 : 200; Jackson ImmunoResearch, West Grove, PA, USA) and then incubated with a combination of Alexa Fluor 488‐conjugated strepavidin and Alexa Fluor 568‐conjugated goat anti‐rabbit immunoglobulins, each for 90 min at RT (dilution 1 : 200; Molecular Probes, Carlsbad, CA, USA). All sections were then washed, mounted on slides, air dried, and cover slipped with Vectashield Fluorescence Mounting Medium (Vector Laboratories, Inc., Burlingame, CA, USA). Controls consisted of the omission of primary and/or secondary antibodies for the different combinations, and these sections consistently failed to exhibit the appropriate immunofluorescence.

Yeo S.‐., Kyle V., Morris P.G., Jackman S., Sinnett‐Smith L.C., Schacker M., Chen C, & Colledge W.H. (2016). Visualisation of Kiss1 Neurone Distribution Using a Kiss1‐CRE Transgenic Mouse. Journal of Neuroendocrinology, 28(11), 10.1111/jne.12435.

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