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    Retrograde Labeling Protocol

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    thumita kumi
    1. Retrograde labeling involves injecting tracers like HRP or dextran into nerves to label neurons that project to specific targets. 2. The protocol involves preparing oxygenated media, dissecting embryos to expose nerves, injecting the tracer into cut nerves or muscle, incubating for labeling, then fixing and processing tissue. 3. Reagents include Tyrode's solution, HRP solution, and dextran rhodamine for labeling, as well as reagents to prepare the black Sylgard plates for dissection and imaging.

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    Retrograde Labeling Protocol

    Uploaded by

    thumita kumi
    25 views2 pages
    1. Retrograde labeling involves injecting tracers like HRP or dextran into nerves to label neurons that project to specific targets. 2. The protocol involves preparing oxygenated media, dissecting embryos to expose nerves, injecting the tracer into cut nerves or muscle, incubating for labeling, then fixing and processing tissue. 3. Reagents include Tyrode's solution, HRP solution, and dextran rhodamine for labeling, as well as reagents to prepare the black Sylgard plates for dissection and imaging.

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    1. Retrograde labeling involves injecting tracers like HRP or dextran into nerves to label neurons that project to specific targets. 2. The protocol involves preparing oxygenated media, dissecting embryos to expose nerves, injecting the tracer into cut nerves or muscle, incubating for labeling, then fixing and processing tissue. 3. Reagents include Tyrode's solution, HRP solution, and dextran rhodamine for labeling, as well as reagents to prepare the black Sylgard plates for dissection and imaging.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    25 views2 pages

    Retrograde Labeling Protocol

    Uploaded by

    thumita kumi
    1. Retrograde labeling involves injecting tracers like HRP or dextran into nerves to label neurons that project to specific targets. 2. The protocol involves preparing oxygenated media, dissecting embryos to expose nerves, injecting the tracer into cut nerves or muscle, incubating for labeling, then fixing and processing tissue. 3. Reagents include Tyrode's solution, HRP solution, and dextran rhodamine for labeling, as well as reagents to prepare the black Sylgard plates for dissection and imaging.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    Retrograde Labeling

    Dasen Lab

    Retrograde labeling / Backfills (HRP, dextran, etc.)

    1. Aerate media using 95% oxygen 5% CO2 at room temp by placing bubblestone
    attached to tubing in container with media. Use Tyrode’s solution or D-MEM/F-12
    with 12mM HEPES and L-Glu, without phenol red.

    2. Prepare a black Sylgard plate containing media under heat lamp to 32oC
    (adjusted by changing lamp position) with aeration.

    3. Remove embryo and place in media on a Sylgard plate, chop off head. Save
    some tissue for genotyping if using mice. If you have multiple embryos, leave on
    ice in media.

    4. Dissect embryo in media and remove skin above muscle. Pressure inject HRP
    or DRA using aspirator tube assembly (Sigma) with pulled injection needle.
    Alternatively, cut nerve with Bioscissors (visualized by GFP) and inject onto the
    stump.

    5. Expose spinal cord by dorsal laminectomy (not necessary for mice e13.5 or
    younger or st29 or younger chicks).

    6. Transfer embryo to under lam. Pin embryo to plate. (Pins are from Fine Science
    Tools (FST)).

    7. Incubate under lamp for 4-6 hours, adding fresh Media every 30-40 minutes.
    Monitor temperature frequently.

    8. Fix in 4% PFA 2-3 hours, wash in PBS (3-4X) and sucrose overnight.
    Retrograde Labeling
    Dasen Lab

    Reagents:
    Tyrode’s Solution
    139 mM NaCl
    3 mM KCl
    17 mM NaHCO3
    12 mM Glucose
    3 mM CaCl2
    1 mM MgCl2

    10X NaCl/KCl/NaHCO3 (1L)


    81.2g NaCl (58.44 g/mol)
    2.24g KCl (74.55 g/mol)
    14.28g NaHCO3 (84.01 g/mol)

    10X MgCl2/CaCl2 (1L)


    1.764g CaCl2 (2H20) (147 g/mol)
    0.814g MgCl2 (6H20) (203.31 g/mol)

    1M glucose (200 ml)


    36.03g dextrose (180.16 g/mol) in ddH20

    For 500mL Tyrodes:


    - add 50ml of 10X NaCl/Cl/NaHCO3, 6ml glucose to 400ml ddH20
    - bubble oxygen in solution for 5 min
    - add 50ml 10X Mg/Cl

    Black Sylgard plate (best for seeing muscles)


    Sylgard 184 Silicone Elastomer Kit (Fisher)
    Prepare 50ml (45 ml + 5 ml curing reagent) per 10 cm plate
    Add charcoal or black ink to darken
    Mix thoroughly and poor into plate
    Cure under vacuum (may take 3 days if using charcoal)

    HRP solution
    Make 10% Lysophosphatidyl choline (Sigma L4129) in PBS (100mg in 1ml PBS)
    Dissolve 100mg HRP (Roche cat# 814 407) in 450 ul PBS on ice add 50 ul 10% Lysophosphatidyl
    choline and vortex.
    Aliquot in small volumes (25-50ul and freeze at –70C)

    Dextran Rhodamine (DRA) (3000 MW lysine fixable Molecular Probes D-3308)


    Make 15% solution by dissolving in ddH20.
    Aliquot and freeze.

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