DNA probe signals were captured by two-steps system detection of biotin-labeled probe with avidin–AlexaFluor488 (Molecular Probes; dilution 1:100; final concentration: 10 µg/ml) and anti-avidin-FITC (Sigma; dilution 1:100; final concentration: 20 µg/ml), and two-steps system detection of digoxigenin-labeled probe with anti-digoxigenin–rhodamine produced in sheep (Roche; dilution 1:100; final concentration: 2 µg/ml) and secondary antibody donkey anti sheep-TRITC (Jackson ImmunoResearch; dilution 1:100; final concentration: 10 µg/ml). DNA was counterstained using Hoechst 33342 (Molecular Probes), and samples were mounted with Vectashield (Vector Laboratories).
Avidin alexa fluor 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Avidin-Alexa Fluor 488 is a conjugate of the protein avidin and the fluorescent dye Alexa Fluor 488. It is designed for use in various bioanalytical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti digoxigenin rhodamine, by Roche (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Anti avidin fitc, by Merck Group (1 mentions)
Donkey anti sheep tritc, by Jackson ImmunoResearch (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Melittin, by Merck Group (1 mentions)
1 palmitoyl 2 oleoly sn 3 glycero phosphocholine popc, by Avanti Polar Lipids (1 mentions)
Axio imager m2p, by Zeiss (1 mentions)
Dig nick translation mix, by Roche (1 mentions)
Isis v5, by MetaSystems (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Neurobiotin, by Vector Laboratories (1 mentions)
Streptavidin alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Ab13970, by Abcam (1 mentions)
Anti synorf1 3c11, by Developmental Studies Hybridoma Bank (1 mentions)
Mw 3000, by Thermo Fisher Scientific (1 mentions)
D3308, by Thermo Fisher Scientific (1 mentions)
7 protocols using avidin alexa fluor 488
1
Two-step DNA Probe Visualization
2
Hybridization Protocol for Gene Status Detection
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For the hybridization procedure, sections were deparaffinized and hydrated in a series of alcohol. Subsequently, digestion with pepsin and tissue permeabilization with sodium thiocyanate at 80°C was performed. The sections were equilibrated in 50% formamide/2X saline-sodium citrate (SSC), pre-hybridized in 45°C, denatured in 80°C and hybridized for 40 h. In brief, post-hybridization procedures were performed by incubation of the sections in 2X SSC and 0.1X SSC in 60°C for 2×10 min. Detection of the biotin-conjugated probes was performed by incubation with avidin-Alexa Fluor® 488 (Molecular Probes, Life Technologies, Inc., Eugene, OR, USA) and anti-avidin-fluorescein-isothiocyanate (Sigma Aldrich, St. Louis, USA) antibodies. For visualization of the nuclei, the sections were stained with Hoechst (Molecular Probes, Life Technologies, Inc.). The specimens were examined under a Nikon Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan). To assess the gene status in each section, 200 cells were counted (magnification, ×600).
3
Immunohistochemistry of CB1 Receptor
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Tissue sections were incubated at 4°C for 48 h with rabbit anti‐CB1 receptor polyclonal antibody directed against the last 15 amino acids of rat CB1 receptor (1:2000) (Bodor et al., 2005 (link)) in PBS containing 0.2% Triton X‐100, 2.5% BSA, and 10% NGS, kindly supplied by Dr. K. Mackie (Indiana State University, USA). Then, sections were washed in PBS containing 0.2% Triton X‐100 and incubated with biotinylated goat anti‐rabbit IgG (1:200, cat no. BA‐1000, Vector Laboratories, Burlingame, CA, USA) for 1 h in the dark at room temperature. Subsequently, sections were incubated with Avidin Alexa Fluor 488 (1:1000, cat no. A‐21370, Molecular Probes, USA) for 1 h in the dark at room temperature and then rinsed and mounted on slides using VectaShield anti‐fade mounting media (Vector Inc.).
Standard control experiments were performed by omitting either the primary or secondary antibody and yielded no cellular labelling (data not shown). All the diluting buffers and the final antibody dilutions were used only once.
Standard control experiments were performed by omitting either the primary or secondary antibody and yielded no cellular labelling (data not shown). All the diluting buffers and the final antibody dilutions were used only once.
4
Lipid and Peptide Preparation Protocol
5
Two-Color FISH Methodology for Genomic Analysis
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Two-color FISH was performed according to our standard protocol [18 (link)] using differently labeled combinations of two or three probes. The probes were labeled by nick-translation either with biotin or digoxigenin using the BIO- or DIG-Nick Translation Mix (Roche, Basel, Switzerland), respectively. Biotin-labeled probes were detected with avidin-Alexa Fluor 488 (Invitrogen) and dig-labeled probes with anti-digoxigenin-Rhodamine (Roche). The results were analyzed with a motorized fluorescence microscope Axio Imager M2p (Zeiss, Jena, Germany), equipped with the Isis v5.2 (MetaSystems GmbH, Altlußheim, Germany) software package for FISH analysis. A minimum of 20 metaphases were captured and analyzed for each experiment.
6
Visualizing Astrocytic and Oligodendrocytic Gap Junctions
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Gap junctional astrocytic and oligodendrocytic networks were visualized as described earlier (Langer et al., 2012 (link); Griemsmann et al., 2015 (link); Augustin et al., 2016 (link)). Astrocytes and oligodendrocytes were tracer- and dye-filled while patch-clamping for 20-130 min. In most experiments, intracellular solution contained a cocktail of the gap junction-permeable tracer neurobiotin (1%, Vector Laboratories, Inc.) and the gap junction-impermeable dye alexa fluor 568 (100 μM, Invitrogen). neurobiotin was identified using avidin alexa fluor 488 (50 μg/ml, Invitrogen; Langer et al., 2012 (link); Augustin et al., 2016 (link)).
In experiments using PLP-GFP mice, the intracellular solution contained biocytin (0.5%). Biocytin was labeled with streptavidin alexa fluor 647 (1:600, Molecular Probes). Simultaneously, PLP-GFP signal was immunohistochemically enhanced (Augustin et al., 2016 (link)). The primary antibody (chicken anti-GFP, ab13970, Abcam) was diluted 1:500 in 0.1% triton X-100 and 2% NGS (Millipore). The secondary antibody (goat anti-chicken alexa fluor 488, A-11039, Invitrogen) was diluted 1:500.
In experiments using PLP-GFP mice, the intracellular solution contained biocytin (0.5%). Biocytin was labeled with streptavidin alexa fluor 647 (1:600, Molecular Probes). Simultaneously, PLP-GFP signal was immunohistochemically enhanced (Augustin et al., 2016 (link)). The primary antibody (chicken anti-GFP, ab13970, Abcam) was diluted 1:500 in 0.1% triton X-100 and 2% NGS (Millipore). The secondary antibody (goat anti-chicken alexa fluor 488, A-11039, Invitrogen) was diluted 1:500.
7
Multimodal Neuroanatomical Tracing in Insects
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Three different staining techniques were used. First, unilateral antennal backfills (right antenna) were performed in eight females, by means of the neuronal tracer neurobiotin (NB, Vector Laboratories), revealed by streptavidin conjugated with the fluorochrome Cy3 (Jackson Immu-noResearch Europe). Second, NB was used as a backfilled tracer, and visualized with avidin-Alexa Fluor 488 (Invitrogen, Molecular Probes) (n = 8 males, n = 4 females). In some specimens (n = 4 males, n = 2 females) we added immunohistochemical labeling with the primary antibody anti-synapsin (anti SYNORF1 3C11, Developmental Studies Hybridoma Bank, University of Iowa, USA), in order to visualize brain areas with high synaptic density (such as the glomeruli in the ALs). We incubated with a goat anti-mouse secondary antibody conjugated with Alexa Fluor 546 (Invitrogen, Molecular Probes). Cell nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI). Third, bilateral or unilateral (right) antennal backfills were performed using the neuronal tracer tetramethylrhodamine-conjugated dextran amine (TMR-DA; Molecular Probes, D3308, MW 3000) (n = 3 males and n = 3 females).
Except otherwise stated, chemicals were purchased from Sigma-Aldrich.
Except otherwise stated, chemicals were purchased from Sigma-Aldrich.
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