Cell cultures were harvested after exposure to colcemid for 4 hours and chromosome preparations were obtained according to standard cytogenetic methods. However, for interphase analysis, samples were produced without colcemid treatment. Cells were spread onto slides and were then denatured in 70% formamide in 2× SSC at 70°C for 5 minutes. The probes were prepared using purified DNA from RP11-265B8 and RP11-876N18 BAC clones, which were labelled by nick translation with biotin and digoxigenin, respectively, according to the manufacturer’s instructions (Roche). The labelled DNAs were ethanol precipitated together with Cot-1 human DNA (Roche) and resuspended in 10 μl of hybridisation buffer (Sigma). The probes were incubated at 65°C for 10 minutes, followed by preannealing at 37°C for 10 minutes. Hybridisation was carried out at 37°C overnight followed by washing with 2× SSC for 5 minutes. The RP11-265B8 probe was detected with Avidin D-Texas Red, biotinylated anti-Avidin D and Avidin D-Texas Red (Vector Laboratories). The RP11-876N18 was detected with mouse anti-digoxigenin antibody (Sigma-Aldrich) followed by rabbit anti-mouse FITC and anti-rabbit FITC (Sigma-Aldrich). The slides were mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA, USA) containing DAPI counterstain.
Texas red avidin d
Manufactured by Vector Laboratories
Sourced in United States, Germany
Texas Red Avidin D is a fluorescently labeled protein used as a detection reagent in various bioassays and research applications. It binds strongly and specifically to biotin, allowing for the visualization and identification of biotinylated molecules. This product provides a stable and reproducible labeling solution for researchers.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (3 mentions)
Hybridisation buffer, by Merck Group (2 mentions)
Cot 1 human dna, by Roche (2 mentions)
Anti rabbit fitc, by Merck Group (2 mentions)
Mouse anti digoxigenin antibody, by Merck Group (2 mentions)
Rabbit anti mouse fitc, by Merck Group (2 mentions)
Biotinylated anti avidin d, by Vector Laboratories (2 mentions)
Biotinylated griffonia, by Vector Laboratories (2 mentions)
Vectashield mounting media for fluorescence, by Vector Laboratories (2 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (2 mentions)
Texasred avidin, by Merck Group (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Digoxigenin, by Roche (1 mentions)
Donkey anti rat cy3, by Jackson ImmunoResearch (1 mentions)
Fluorescein avidin d, by Vector Laboratories (1 mentions)
Anti iba1, by Abcam (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Dm5000b microscope, by Leica camera (1 mentions)
Axio observer, by Zeiss (1 mentions)
Dq red bsa, by Thermo Fisher Scientific (1 mentions)
12 protocols using texas red avidin d
1
Interphase FISH Analysis of Genomic Regions
2
Fluorescence In Situ Hybridization Assay
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Cell cultures were harvested after exposure to Colcemid for 4 hours and chromosome preparations were obtained according to standard cytogenetic methods. For interphase analysis, samples were produced with the exception of Colcemid treatment. Cells were spread onto slides and were then denatured in 70% formamide in 2× SSC at 70°C for 5 minutes. The probes were prepared using purified DNA from RP11-265B8 and RP11-876N18 BAC clones, which were labeled by nick translation with biotin and digoxigenin respectively, according to the manufacturer's instructions (Roche, Mannheim, Germany). The labeled DNAs were ethanol precipitated together with Cot-1 human DNA (Roche) and resuspended in 10 µl of hybridisation buffer (Sigma). The probes were denatured by incubating at 65°C for 10 minutes, followed by preannealing at 37°C for 10 minutes. Hybridisation was at 37°C overnight followed by washing with 2× SSC for 5 minutes. The RP11-265B8 probe was detected with Avidin D-Texas Red, biotinylated anti-Avidin D and Avidin D-Texas Red (Vector Laboratories). The RP11-876N18 was detected with mouse anti-digoxigenin antibody (Sigma-Aldrich) followed by rabbit anti- mouse-FITC and anti- rabbit-FITC (Sigma-Aldrich). The slides were mounted in VECTASHIELD (Vector Laboratories, Burlingame, CA, USA) containing DAPI counterstain.
3
Immunofluorescent Analysis of Tumor Angiogenesis and Macrophages
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Freshly isolated murine tumor grafts were fixed and stained with a primary antibody to CD34 (RAM34; dilution 1:50; BD Biosciences) and detected by donkey anti-rat–Cy3 (1:10.000 dilution; Jackson Immuno). Double immunofluorescence was performed for detecting pSTAT3(Y705) in TAMs. Briefly, tumor sections were stained with the pSTAT3(Y705) antibody described above overnight and detected with a secondary biotinylated anti-rabbit (1:500 dilution; Vector Laboratories) and Fluorescein Avidin D (1:200 dilution; Vector Laboratories). To detect macrophages, tumor sections were stained with anti-Iba1 (1:200 dilution; Abcam catalog no. ab5076) for 1 h followed by a secondary biotinylated anti-goat (1:500 dilution; Vector Laboratories) and Texas Red Avidin D (1:200 dilution; Vector Laboratories). Sections were mounted with ProLong Gold antifade with DAPI (Molecular Probes). Fluorescence images were captured by using a Leica DM5000 B microscope. Image analysis was performed by using ImageJ software.
4
Nuclei Isolation and 3D FISH Imaging
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The nuclei were isolated from leaves of 35-day-old rosettes grown under a 16-h/8-h photoperiod. Nuclei extraction and embedding in acrylamide gel pads on the slide were done as described [42 (link)]. Centromeric and 45S rDNA repeats were detected by FISH using pAL1 and pTA9 to generate DNA probes, respectively [39 (link)]. FISH was done as described [42 (link)] with the following labeling kits and fluorescent immunolabeling reagents: DIG-Nick (Sigma Aldrich, 11745816910), mouse IgG anti-DIG (1:250, Sigma Aldrich, 11333062910), goat IgG anti-mouse IgG~Alexa 488 (1:200, Life Technologies, A11001); Biotin-Nick translation kit (Sigma Aldrich, 11745824910), Biotinylated Anti-Avidin D (1:250, Vector Labs, BA-0300), and Texas Red Avidin D (1:1000, Vector Labs, A-2006). The nuclei were counterstained for DNA with DAPI in Vectashield (Vector Laboratory). FISH signals in 3D nuclei were imaged using Stimulated emission depletion (STED) microscopy (Leica SP8R WL 3xSTED, Leica microsystems, Germany).
5
Retinal Vascular and Neuronal Imaging
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Retinas were dissected and permealized for 15 minutes with 0.3% Triton X-PBS then stained overnight at 4 °C with isolectin; biotinylated griffonia (bandeiraea) simplicifolia lectin I (GSL I, BSL I), (Vector Labs, CA, USA; 1% in 5% normal goat serum in 0.3% Triton X-PBS) followed by incubation with secondary antibody; Texas red® avidin D (Vector labs, CA, USA; 0.5% in 5% normal goat serum in 0.3% Triton X-PBS). Retinal perfusion was assessed by intraperitoneal injection of FITC-dextran (Mol. WT. 2,000,000, Sigma, 50 mg mL−1, 100 μL/pup) 30 minutes prior to sacrifice as described previously23 (link). Lectin-stained and/or FITC-perfused retinas were flat-mounted onto Super-frost/Plus microscope slides (Fisher Scientific, MA, USA) with the photoreceptor side facing down and imbedded in Vectashield mounting media for fluorescence (Vector Labs, CA, USA). Slides were photo-micrographed at 5X using a Zeiss AxioObserver.Z1. Images were assembled into a single file using photoshop software (CS6, Adobe system incorporated). CDO and RNV were quantified as previously described24 (link).
6
Fluorescent Uptake Assays in Drosophila
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For TexasRed-Avidin and DQ Red BSA uptake assays, L3 larval proventriculi with garland nephrocytes were prepared in cold Shields and Sang M3 insect medium (Merck, Darmstadt, Germany, S8398) and incubated in 0.1 mg/mL TexasRed-Avidin D (Vector Laboratories, A-2006) or 10 µg/mL DQ Red BSA (Thermo Fisher Scientific, Waltham, MA, USA, D12051) containing M3 for 5 min at RT, rinsed 3 times, and incubated in M3 for 30 min where stated, then fixed with 4% formaldehyde in PBS (50 min at RT). Samples were washed 3 × 10 min in PBS, stained with DAPI and mounted as described before. For LysoTracker staining, fat bodies were dissected in cold PBS and incubated in LysoTracker Red DND-99 (1:1000 in PBS; Thermo Fisher Scientific, L7528) for 1 min at RT. Samples were rinsed 3 times, mounted in 80% glycerol in PBS containing DAPI, and photographed immediately. Salivary glands were dissected in cold PBS, then fixed with 4% formaldehyde in PBS (5 min at RT), and mounted in 80% glycerol in PBS containing DAPI.
7
Retinal Vessel Staining with Lectins
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3 or 7 days post intravitreal injection of MSCs, mice were euthanized in CO2 chamber (2% flow rate for 5 min), followed by cervical dislocation. Eyes were enucleated and fixed in 2% paraformaldehyde overnight. Retinas were dissected and permeabilized for 15 min with 0.3% Triton X-PBS then stained overnight at 4 °C with isolectin B4; biotinylated griffonia (bandeiraea) simplicifolia lectin I (GSL I, BSL I), (Vector Labs, Burlingame, CA, USA; 1% in 5% normal goat serum in 0.3% Triton X-PBS), followed by incubation with secondary antibody; Texas red® avidin D (Vector labs; 0.5% in 5% normal goat serum in 0.3% Triton X-PBS). Lectin-stained retinas were flat-mounted onto Superfrost/Plus microscope slides (Fisher Scientific, Waltham, MA, USA) with the photoreceptor side facing down and imbedded in Vectashield mounting media for fluorescence (Vector Labs). Slides were photo-micro-graphed at 40x using a Zeiss Axio Observer Z1 (Carl Zeiss Vision Inc.,Thornwood, NY, USA).
8
Immunohistochemical Staining of Kidney Tissue
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Kidneys were fixed with formalin and embedded in paraffin before they were sectioned. Each section was incubated in xylene for 8 minutes before being rehydrated in decreasing concentrations of ethanol. Sections were then washed twice with ddH2O and placed into 1x PBS. Antigen retrieval was achieved by microwaving sections in 600 mL of ddH2O + 5.6 mL of Vector Antigen Retrieval solution concentrate. Sections were then washed with 1x PBS for 5 minutes at room temperature with shaking. Sections were then blocked in 5% normal goat serum in 1x PBS for 1 hour at 37°C. The sections were then stained with biotinylated goat anti-mouse total Ig (Southern Biotech) at a dilution of 1:50 in 5% goat serum in 1x PBS for 1 hour at 37°C. Slides were washed 3 times for 5 minutes with 1x PBS and then incubated with Texas-Red Avidin D (Vector Laboratories) for 1 hour at 37°C. Slides were then washed 3 times with 1x PBS, and VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) was added before the addition of a coverslip and the imaging of the slide.
9
Western Blot Antibody Optimization Protocol
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Monoclonal anti-Caspase-12 antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-cleaved caspase-3, anti-PUMA, anti-HistonH2A, anti-AIF, anti-RIP1, anti-RIP3, anti-MLKL, anti-p53, anti-COX IV and anti-Lamin B were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-RPE65 (specific for RPE cells) was from Abcam (Cambridge, MA, USA). The preparation and use of FITC-labeled anti-MCMV EA and biotin-anti-EA has been described previously [24 (link)]. Anti-β-actin was from Sigma-Aldrich Corp. (St. Louis, MO, USA). Texas red avidin D, DyLight 594 goat anti-rabbit IgG antibody and DyLight 488 horse anti-mouse IgG antibody were from Vector Laboratories, Inc. (Burlingame, CA, USA). Goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). BCA assay kit, NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (78833) and Mitochondria Isolation Kit for Tissue (89801) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). TUNEL assay kit was from Roche (Indianapolis, IN, USA).
10
Adenovirus-Mediated IGFBP-5 Expression in Lung Fibroblasts
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Human lung fibroblasts were seeded in chamber slides (BD Biosciences, Bedford, MA) and infected with replication-deficient adenoviruses encoding IGFBP-5 or NLS-mutant IGFBP-5 for 72 hours. Cells were fixed with 2% of paraformaldehyde for 15 minutes, permeabilized with 0.1% TritonX-100 for 15 minutes, blocked with 5% of goat normal serum for one hour, and incubated with anti-IGFBP-5 antibody (Gropep, Thebarton, SA, Australia) overnight at 4°C. Slides were washed with 1X PBS three times, incubated with biotinylated secondary antibody for one hour at room temperature, followed by Texas Red Avidin D (Vector Labs, Burlingame, CA). Hoechst (Sigma, St Louis, MO) was used to identify nuclei. Images were taken on an Olympus Provis 3 microscope (Olympus Corporation, Melville, NY).
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