The chorda tympani and greater superficial petrosal nerves (gustatory branches of cranial nVII) were exposed in anesthetized mice and cut immediately distal to the tympanic bulla. Crystals of fluorescent dextran were placed on the cut proximal end as previously described21 (link). Dyes selected were lysine fixable 3 kDa dextrans, conjugated to fluorescein or tetramethylrhodamine (ThermoFisher D3306, D3308). After 5–7 h to allow dextran to accumulate in the neuronal soma, mice were fixed by perfusion and ganglia were harvested and processed for immunohistochemical analyses as described above.
D3306
Manufactured by Thermo Fisher Scientific
Sourced in United States
The D3306 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for sample preparation and analysis tasks in a laboratory setting. The core function of the D3306 is to provide accurate and reliable results for various analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
D3308, by Thermo Fisher Scientific (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Fitc dextran, by Thermo Fisher Scientific (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Wga af647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Carl Roth (1 mentions)
Synergy h1mf, by Agilent Technologies (1 mentions)
Bl539a, by Biosharp (1 mentions)
Kwik sil, by World Precision Instruments (1 mentions)
7 protocols using d3306
1
Tracing Gustatory Nerves in Mice
2
Dextran Microinjection into Chicken Embryo DM
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Dextran injections into the DM were done using 3000 MW dextran conjugated to fluorescein (ThermoFisher D3306) at a concentration of 10 mg/mL in 1X PBS with Fast Green dye added to better visualize the solution during injection. This mixture was loaded into fine pulled glass capillary needles. A microinjector with a foot pedal was set to 5 psi for 200 ms. Chicken embryos at the desired stage were prepared by removing the vitelline membrane. Injections were done to the right DM only, because embryos lie on their left sides from HH18 onwards and only the right DM is accessible for injection. With the anterior/posterior axis of the embryo perpendicular to the needle and with the needle at a 25° angle, the body wall was gently pulled back so the needle could access the right side of the DM. The needle was gently pressed into the tissue until the embryo moved slightly from the force. Then the foot pedal was pressed once to inject. Embryos were allowed to continue incubating for about two hours, then embryos were collected and fixed in 2% PFA overnight at 4°C. To screen for embryos with quality injections, embryos were cryo-embedded and sectioned. Any embryos with visible damage to the DM in these sections were excluded from further analysis.
3
Visualizing Endosomal Escape with LLO Y406A
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To reveal endosomal escape, RT4 cells were co-incubated with 0.5 µM LLO Y406A and 0.5 mg/mL FITC-dextran (3kDa, D3306, Thermo Fisher Scientific, Waltham, MA, USA) for 2 h, washed in control medium, and cultured in control medium for 1 h. Alternatively, RT4 cells were co-incubated with 5.0 µM mCherry-LLO Y406A and FITC-dextran following the same protocol. Then cells were then fixed in 4% formaldehyde in PBS (pH 7.4) at 22 °C for 15 min and mounted in Vectashield with DAPI (4,6-diamidino-2-phenylindole) for nuclear staining (Vector Laboratories). Additionally, LysoTracker Red DND-99 (L7528, Molecular Probes Life Technologies, Waltham, OR USA) was used. In brief, RT4 cells were incubated with 50 nM LysoTracker at 37 °C for 45 min. Cells were then washed and LLO Y406A was added to a final concentration of 0.5 µM for 2 h and washed with control medium. One h later, cells were fixed in 4% formaldehyde in PBS at 22 °C for 15 min and mounted in Vectashield with DAPI. Cells were analyzed using a 20×/NA 0.75 and 63×/NA 1.4 objectives on a fluorescence microscope AxioImager Z.1 with an Apotome device for optical section generation (Zeiss).
4
Fluorescent Dextran Labeling Protocol
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Fluorescent dextrans of 3 kDa (D3306) and 500 kDa (D7136) were purchased from Thermo Fisher Scientific Inc (USA).
5
Assessing Tissue Slice Viability
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A Dextran uptake assay in combination with ryanodine receptor (RyR) immunofluorescence was performed to assess viability of the tissue slices according to a published method (Pfeuffer et al., 2023 (link)). Briefly, dextran (3 kDa) conjugated to FITC (Thermo Fisher, D3306) at a concentration of 2 mg/mL was incubated for 10 min at 37°C and 5% CO2 under continuous rocking on the MyoDish culture system platform. The slices were immediately fixed with 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min and then washed three times with PBS for 5 min each. Subsequently, slices were incubated with primary antibody against cardiac RyR (IgG1, mouse, C3-33, Thermo Fisher, Braunschweig, Germany) 1:200 in blocking solution (BS: 5% NGS, 5% BSA, 0.25% Triton-X in PBS) for 4 h at RT or overnight at 4°C, washed three times with PBS for 5 min and incubated with the secondary antibody goat anti-mouse IgG1 AF-555 (Thermo Fisher A-21127) 1:400 in BS for 3 h at RT. After washing, the slices were incubated with wheat germ agglutinin (WGA-AF-647 Thermo Fisher W32466) 40 μg/mL and DAPI (3665, Roth, Karlsruhe, Germany) 1.67 μg/mL in PBS for 3 h. The slices were mounted with Fluoromount G (00-4958–02, Thermo Fisher; F4680 Sigma-Aldrich, Darmstadt, Germany) on a glass microscope slide, covered with a coverslip and equilibrated for 3–7 days at 40%–45% humidity.
6
Blood-Brain Barrier Permeability Assessment
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BBB integrity in mice was estimated with BBB permeability analysis. In BBB permeability assessment, a single dose of 3kDa-Dextran (3kDa-Dex, either labeled with Alexa fluor −488 or −555, Invitrogen D3306, Invitrogen D3308) was intravenously injected to mice (10 mg/kg) at 90 min before sacrifice. After collection of peripheral blood, eyeballs of mice were dissected and perfusion with PBS (10 ml) then 4% paraformaldehyde (Biosharp BL539A, 10 ml) was performed. Two eyeballs of each recipient were ground in 500ul PBS. Fluorescent intensity of the 3kDa-Dextran in plasma and the eyeball extract was measured with a 96-well plate reader (Biotek Synergy H1MF). Extravasation index of 3kDa-Dextran in brain parenchyma of the recipients was calculated as the ratio of extra- to intra-vessel 3kDa-Dextran MFI (Image J, NIH) which was further normalized to that of the MOCK group.
7
Fluorescent Labeling of Gustatory Nerves
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