Transplantation assays were performed as previously described (Aman and Piotrowski, 2008 (link)). Donor embryos were injected with 5% Alexa-568 and 3% lysine-fixable biotinylated-dextran (Invitrogen, USA) at the one cell stage. Tg(foxd3:gfp) or Tg(clndB:lyngfp) wild type donors were used to test rescue of erbb2 mutant hosts. Tg(clndB:lyngfp) donors were used to test rescue of nrg1-3z26 mutant hosts. Host embryos were screened for lateral line or Schwann cell clones at 24 hr post fertilization (hpf). Embryos were imaged at 48 hpf to record the extent of the transplanted clone. To identify mutant hosts or donors, neuromasts were counted after DASPEI staining at 4 days post fertilization (dpf). Both transplanted and untransplanted sides of the hosts were imaged with a Zeiss LSM 510 or 780 confocal microscope at 20X. For low magnification images, individual images were stitch together using ImageJ.
Biotinylated dextran
Manufactured by Thermo Fisher Scientific
Sourced in United States
Biotinylated dextran is a polymer used as a reagent in various biotechnological applications. It contains biotin molecules covalently attached to the dextran backbone, enabling specific interactions with streptavidin or avidin proteins. This reagent can be utilized in techniques such as affinity chromatography, immunoassays, and histological staining, where the biotin-streptavidin/avidin interaction is employed.
Automatically generated - may contain errors
Lab products found in correlation
Anti claudin 5, by Thermo Fisher Scientific (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Streptavidin fitc, by Thermo Fisher Scientific (1 mentions)
Actin binding phalloidin alexa350, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Tetramethylrhodamine dextran, by Thermo Fisher Scientific (1 mentions)
D3307, by Thermo Fisher Scientific (1 mentions)
D 7135, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Cas 30525 89 4, by Santa Cruz Biotechnology (1 mentions)
S32354, by Thermo Fisher Scientific (1 mentions)
S32357, by Thermo Fisher Scientific (1 mentions)
Ms 222, by Merck Group (1 mentions)
P6744, by Merck Group (1 mentions)
Vectashield hardset, by Vector Laboratories (1 mentions)
6 protocols using biotinylated dextran
1
Transplantation Assays for Zebrafish Mutants
2
Immunofluorescence Analysis of Claudin-5 and IgG
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Immunofluorescence for claudin 5 and IgG were performed on 40 μm cryo
sections with anti-claudin 5 (1:200, Thermo Fisher), anti-mouse IgG antibody
(1:200, Molecular Probes) and anti-ZO1 (1:400, Thermo Fisher), respectively.
For double-fluorescence labeling for IgG and isolectin B4, cryo sections (10
μm) were first incubated with anti-biotinylated isolectin B4 antibody
(1:100, Sigma) at room temperature for 1 h. Subsequently, they were
incubated with secondary antibody, streptavidin-conjugated Alexa 488 at room
temperature for 30 min. After being washed in PBS, they were then incubated
with goat anti-mouse IgG antibody coupled to Alexa Fluor 594 (Molecular
Probes) at a dilution of 1:200 for 30 min. Finally, they were examined in an
FSX100 microscope. Trans-cardiac perfusion of E18 embryos was performed with
3 kDa biotinylated dextran (0.15 mg/ml, Invitrogen), tissue sections were
stained with streptavidin Alexa 594 and fluorescence in tissue sections was
quantified by ImageJ software. Perfusions and analysis was done blinded to
genotype.
sections with anti-claudin 5 (1:200, Thermo Fisher), anti-mouse IgG antibody
(1:200, Molecular Probes) and anti-ZO1 (1:400, Thermo Fisher), respectively.
For double-fluorescence labeling for IgG and isolectin B4, cryo sections (10
μm) were first incubated with anti-biotinylated isolectin B4 antibody
(1:100, Sigma) at room temperature for 1 h. Subsequently, they were
incubated with secondary antibody, streptavidin-conjugated Alexa 488 at room
temperature for 30 min. After being washed in PBS, they were then incubated
with goat anti-mouse IgG antibody coupled to Alexa Fluor 594 (Molecular
Probes) at a dilution of 1:200 for 30 min. Finally, they were examined in an
FSX100 microscope. Trans-cardiac perfusion of E18 embryos was performed with
3 kDa biotinylated dextran (0.15 mg/ml, Invitrogen), tissue sections were
stained with streptavidin Alexa 594 and fluorescence in tissue sections was
quantified by ImageJ software. Perfusions and analysis was done blinded to
genotype.
3
Visualizing Intranasal Antigen Deposition in Murine Lungs
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To confirm the presence of i.n.-applied antigens within the respiratory tract, 20 nm NPs (10% from an original concentration of 2%) and soluble antigens (Ova (5 mg/20 µL, Sigma), lysine-fixable biotinylated dextran (10 K MW, Invitrogen)) were i.n. administered to anesthetized mice in a volume of 20 μL (10 μL/ nostril). At 1 h after antigen administration mice were euthanized, lung tissues were excised and snap-frozen in optimum cutting temperature (O.C.T.) freezing medium. Tissue cryosections were fixed in 10% PFA then stained with streptavidin-FITC (fluorescein isothiocyanate) and antibodies specific for Ova and DC marker CD11c (eBioscience). Tissue architecture was highlighted with actin-binding phalloidin-Alexa350 (Invitrogen). Stained tissue sections were imaged with a Leica DM1000B fluorescence microscope and acquired images were analyzed as described previously [34 (link),38 (link),39 (link)]. To get a more detailed view of the tissue, lung sections were also stained with hematoxylin and eosin (H&E) and imaged with an Olympus BX41 microscope equipped with an Olympus DP72 camera using CellSens Standard imaging software.
4
Retrograde Tracing of Zebrafish Motor Neurons
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Retrograde labeling of axial motor neurons was performed in anesthetized zebrafish with 0.03% tricaine methane sulfonate (MS-222, E10521; Sigma-Aldrich) by using dye injections with tetramethylrhodamine-dextran (3,000 molecular weight, D3307; Thermo Fisher) or with biotinylated dextran (3,000 molecular weight, D7135; Thermo Fisher) into all muscles or into specific muscle fiber types (slow, intermediate, or fast) in myotomes 14–16 (21 (link), 42 (link), 43 (link)). All of the injected animals were allowed to recover for 5 h to overnight to help the retrograde transport of the tracer.
5
Staining and Flow Cytometry of pHLA-E
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To stain peptide-HLA-E (pHLA-E) with CD94/NKG2x tetramers, each biotinylated receptor was mixed with streptavidin coupled to AlexaFluor647 (SAV-647, made in-house) at a 5:1 ratio and incubated for 5 min on ice. Yeast were stained with 500 nM tetramer for 2 h at 4 °C in the dark with rotation. Then, the yeast were washed twice with FACS buffer (0.5% BSA and 2 mM EDTA in 1x PBS) before analysis via flow cytometry (Accuri C6 flow cytometer, BD Biosciences; Franklin Lakes, New Jersey). Dextramer staining was performed as with tetramers, excepting CD94/NKG2x protein was incubated with 25 nM biotinylated dextran 70,000 MW (Thermo Fisher) for five minutes before addition of SAV-647. Tetramer staining coupled an anti-streptavidin antibody was performed as with tetramers, excepting 250 nM PE anti-streptavidin antibody (Biolegend) was added to tetramer solution before adding to yeast.
Antibody staining was performed on yeast washed into FACS buffer, with antibody at a 1:50 volume ratio. Yeast incubated with antibody for at least 20 min and excess antibody was removed by washing with FACS buffer. Staining was assessed on the Accuri C6 flow cytometer. Antibody clone 3D12 was used for anti-HLA-E staining.
Antibody staining was performed on yeast washed into FACS buffer, with antibody at a 1:50 volume ratio. Yeast incubated with antibody for at least 20 min and excess antibody was removed by washing with FACS buffer. Staining was assessed on the Accuri C6 flow cytometer. Antibody clone 3D12 was used for anti-HLA-E staining.
6
Retrograde Labeling of Zebrafish Spinal Neurons
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Zebrafish were anesthetized in 0.03% tricaine methane sulfonate (MS-222, Sigma-Aldrich, E10521). Retrograde labeling of descending spinal cord neurons located in spinal segments 1–3 was achieved through dye injections with biotinylated dextran (3000 MW; ThermoFisher, D7135) into segment 16 or 17. Animals were kept alive for at least 24 h after injection to allow retrograde transport of the tracer, deeply anesthetized with 0.1% MS-222, and the spinal cords were dissected and fixed in 4% paraformaldehyde (PFA) and 5% saturated picric acid (Sigma-Aldrich, P6744) in phosphate-buffered saline (PBS; 0.01 M, pH = 7.4; Santa Cruz Biotechnology, Inc., CAS30525-89-4) at 4 °C for 4–10 h. The tissue was then washed extensively with PBS and incubated in streptavidin conjugated to Alexa Fluor 488 (dilution 1:500, ThermoFisher, S32354), Alexa Fluor 555 (1:500, ThermoFisher, S32355), or Alexa Fluor 647 (dilution 1:500, ThermoFisher, S32357) overnight at 4 °C. Primary and secondary antibodies were applied as described in the “Immunohistochemistry” section. After thorough buffer rinses, the tissue was mounted on gelatin-coated microscope slides and cover-slipped with an anti-fade fluorescent mounting medium (Vectashield Hard Set, VectorLabs; H-1400).
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