Mouse kidneys were fixed with the 4% paraformaldehyde solution (pH 7.2) overnight at 4°C, washed with phosphate buffered saline (PBS), cryoprotected with 10% and 20% sucrose at 4°C, embedded in OCT compound (Miles), and frozen in hexane at −80°C. Frozen sections (5 μm thick) were collected onto MAS‐coated slides (Matsunami Glass IND, Ltd.) and air‐dried (Ohkido, Segawa, Yanagida, Nakamura, & Miyamoto, 2003 ; Segawa et al., 2005 ). Serial sections were incubated with primary antibodies overnight at 4°C. Alexa Fluor 488 anti‐rabbit (Molecular Probes) and Alexa Fluor 568 anti‐mouse (Molecular Probes) were used as secondary antibodies for 60 min at room temperature. Thereafter, the sections were mounted with Dapi Fluoromount GTM (Southern Biotech).
Dapi fluoromount gtm
Manufactured by Southern Biotech
Sourced in United States
Dapi Fluoromount GTM is a mounting medium designed for fluorescence microscopy. It is formulated to preserve and protect fluorescent signals, particularly those from DAPI (4',6-diamidino-2-phenylindole) staining.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Mas coated slides, by Matsunami (2 mentions)
Anti mouse alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Mrfp rab5, by Addgene (1 mentions)
Annexin 5 cy5, by Abcam (1 mentions)
Anti actin clone c4, by Merck Group (1 mentions)
Recombinant human trail, by R&D Systems (1 mentions)
Anti bcl 2 n 19, by Santa Cruz Biotechnology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Anti myc 9b11, by Cell Signaling Technology (1 mentions)
Anti caspase 8 1c12, by Cell Signaling Technology (1 mentions)
Anti flag hrp, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Westernbright ecl hrp substrate, by Advansta (1 mentions)
Forskolin, by Bio-Techne (1 mentions)
Kt5720, by Bio-Techne (1 mentions)
Sb202190, by Bio-Techne (1 mentions)
6 protocols using dapi fluoromount gtm
1
Immunofluorescence Staining of Mouse Kidneys
2
Visualizing Endocytic Pathways with Fluorescent Fusion Proteins
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pmRFP-Rab5 was obtained from Addgene (Addgene plasmid 14437). pcDNA4-EGFP-DTX1 and pcDNA4-LAMP1-mCherry were generated as described previously9 (link). To generate pcDNA4-cerulean-c-FLIPL, cerulean fragments were amplified by PCR from Cerulean-GalT (Addgene plasmid 11930) with HindIII/EcoRI and cloned into pcDNA4-c-FLIPL-Myc with HindIII/EcoRI to create a translational fusion construct.
293T cells (1 × 106) transduced with mRFP-Rab5 or LAMP1-mCherry were transfected with cerulean-c-FLIPL and EGFP-DTX1. 24 h after transfection, cells were re-seeded on a 22 × 22 mm glass coverslip and allowed to attach for another 18 h. Cells were fixed in 2% paraformaldehyde in PBS for 15 min at 37 °C and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. Cells were mounted in Dapi-Fluoromount-GTM (SouthernBiotech, Birmingham, AL) and observed under a Zeiss LSM 780 confocal microscope.
293T cells (1 × 106) transduced with mRFP-Rab5 or LAMP1-mCherry were transfected with cerulean-c-FLIPL and EGFP-DTX1. 24 h after transfection, cells were re-seeded on a 22 × 22 mm glass coverslip and allowed to attach for another 18 h. Cells were fixed in 2% paraformaldehyde in PBS for 15 min at 37 °C and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. Cells were mounted in Dapi-Fluoromount-GTM (SouthernBiotech, Birmingham, AL) and observed under a Zeiss LSM 780 confocal microscope.
3
TRAIL-induced Apoptosis Pathway Assay
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Recombinant human TRAIL and anti-His were obtained from R&D Systems (Minneapolis, MN). MG132, propidium iodide (PI), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and anti-Flag-HRP were purchased from Sigma (St. Louis, MO). Rabbit anti-DTX1 polyclonal antiserum against GST-Deltex1 was generated as described9 (link). The following antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA): anti-caspase-3 (H-277), anti-DR4 (H130), anti-DR5 (N-19), anti-Mcl-1 (S-19), and anti-Bcl-2 (N-19). Annexin V-Cy5 was obtained from Biovision (Mountain View, CA). Anti-Myc (9B11), anti-caspase-8 (1C12), and anti-active caspase-3 (D175) were purchased from Cell Signaling (Beverly, MA). Anti-actin (clone C4) and anti-β-tubulin (clone AA2) were purchased from Millipore (Temecula, CA). Anti-human FLIP mAb (NF6) was purchased from AdipoGen (San Diego, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. WesternBright ECL HRP substrate was obtained from Advansta Corporation (Menlo Park, CA). Dapi-Fluoromount-GTM was obtained from SouthernBiotech (Birmingham, AL). Protein G Mag SepharoseTM Xtra was obtained from GE Healthcare (Piscataway, NJ).
4
Endothelial Cell Activation Assay
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KT5720 (PKA inhibitor), PKI 14–22 amide, p53 inhibitors (Pifithrin α and µ), p38 MAP kinases inhibitors (BIRB796 and SB202190), Sphingosine-1-P, Forskolin, and the NF-κB inhibitor BAY11-7085 were all purchased from Tocris (Bristol, UK). For immunostainings, Phalloidin 547 (FP-AZ0330) was from FluoProbes, Interchim, (Montluçon, France), while the FITC-conjugated mouse anti-VE-Cadherin (CD144) antibody (clone 55-7H1, cat no. 580411) was from BD Pharmingen (Franklin Lakes, NJ, USA). The mounting medium (DAPI Fluoromount-GTM (cat no. 0100-20)) was purchased from Southern Biotech (Birmingham, AL, USA). For flow cytometry analysis, all antibodies (BB515 mouse anti-human CD54 (ICAM-1, cat no. 564685), BUV737 mouse anti-human CD106 (VCAM-1, cat no. 565418), PE mouse anti-human CD62P (P-Selectin, cat no. 555524), APC mouse anti-human CD62E (E-Selectin, cat no. 551144)), and the FITC Annexin V/Propidium Iodide apoptosis detection kit were obtained from BD Biosciences (Franklin Lakes, NJ, USA).
5
Immunofluorescence Staining of Rat Tissues
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Rat kidney and intestine tissue samples were fixed in 4% paraformaldehyde solution (pH 7.2) overnight at 4℃. Samples were then washed with phosphate-buffered saline, cryoprotected with 30% sucrose at 4℃, embedded in OCT compound (Miles), and frozen in hexane at -80℃. Frozen sections (5 μm thick) were transferred to MAS-coated slides (Matsunami Glass IND, Ltd.) and air-dried. Frozen sections were incubated with primary antibody overnight at 4℃. Sections were then incubated with Alexa Fluor 488 anti-rabbit or anti-mouse antibody (Molecular Probes) and Alexa Fluor 568 anti-mouse or anti-rabbit antibody (Molecular Probes) for 60 min at room temperature. Sections were mounted with Dapi Fluoromount GTM (Southern Biotech).
6
Validating αvβ3 Expression in U87MG Tumor
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The V 3 expression in U87MG tumor cells was validated in both cell culture and xenograft levels. In brief, U87MG cells were analyzed by flow cytometry with the anti-human α V β 3 antibody (LM609, 1:100 dilution; Millipore). V 3 -Overexpressing HEK293( 3 ) and V 3 -negative HEK293( 1 ) cells (kindly provided by J.-F. Gourvest, Aventis, France) were used as the positive and negative controls, respectively. For U87MG xenograft, α V β 3 expressed by tumor cells and neovascular endothelial cells was validated by immunofluorescence staining with the anti-human α V β 3 antibody and double staining with the anti-mouse CD61 antibody (2C9.G2, 1:50 dilution; BD Biosciences) and the anti-mouse CD31 antibody (MEC 13.3, 1:1000 dilution; BD Biosciences). The slides were mounted with mounting agent (Dapi-Fluoromount-G TM ; SouthernBiotech) containing 4′,6-diamidino-2-phenylindole (DAPI) for nucleus staining. Fluorescence images were acquired with an epifluorescence microscope (Olympus X61).
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