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    • CommentAuthorjakhan66
    • CommentTimeMay 7th 2013 edited
     

     Written by Jannat Khan
    Revised on 05/15/2013

    Material
    Location in the Lom Lab
    Recipe (if needed)

    10X Tricaine solution
    In the non-fix fridge labeled “10x Tricaine”
    Recipe: http://protocols.davidson.edu/comments.php?DiscussionID=86

    Triton X-100 (Sigma-Aldrich, catalog # T9284)
    In the chemical cabinet in a dark glass bottle.

    Phosphate buffered saline (PBS)
    On the shelf in front of the chemical cabinet.
    Recipe: http://www.scientistsolutions.com/t3072-10x+pbs+recipe.html

    4% paraformaldehyde (PFA) (Also referred to as BT-Fix)
    Frozen supplies in the fix freezer. Gently thaw 4% PFA at room temperature the day before planning to fix the zebrafish and store in the fix fridge at 4°C.
    Recipe: http://protocols.davidson.edu/comments.php?DiscussionID
    =16&page=1#item_0

    Glycine (Sigma-Aldrich, catalog # G7126)
    In the chemical cabinet bottle in a white plastic bottle.

    Bovine serum albumin Cohn Analog (Sigma-Aldrich, catalog# A1470)
    In the first shelf of the non-fix fridge door.

    Goat serum (Sigma-Aldrich, catalog # G9023)
    Frozen supplies in plastic tube in the non-fix freezer. Thaw at 37°C.

    Anti-acetylated tubulin (Sigma-Aldrich, catalog # T7451, monoclonal)
    Anti-thyrosine hydroxylase (TH) (Millipore, catalog # AB152, polyclonal)
    Aliquots in white box in small freezer.

    Zn8 (DSHB, concentrate 0.1 mL, monoclonal)
    SV-2 (DSHB, concentrate 0.1 mL, monoclonal)
    Elavl4 (abcam, catalog # ab14369, polyclonal)
    Aliquots on top shelf of the non-fix-freezer.

    Alexa 488 (Invitrogen, catalog # A-11001, goat anti-mouse)
    Inside dark plastic cylinder in non-fix fridge.

    Hoechst Stain (Invitrogen, catalog # H21492)
    In the second shelf of the fix fridge door.

    Day 1:
    Goals: To preserve zebrafish at 72 hours post fertilization.
    Visual: Demonstration of Day 1 and Day 2 (***Note: For this protocol use only 1 X PBS instead of DEPC treated 1 X PBS, which is used when conducting in situ hybridization)
    http://www.youtube.com/watch?v=eCifhnkRGRE

    1. [ ] Remove half of the E3 fish juice from the petri dish with zebrafish.
    ***Note: Use the deep petri dishes to treat the zebrafish.
    2. [ ] Add 10X Tricaine solution to the petri dish so that it is approximately a 50:50 solution of 10X Tricaine and E3 fish juice.
    3. [ ] Wait for embryos to slow their swimming.
    4. [ ] Using a disposable plastic 1 mL pipette suction embryos into a glass vial (8 dram or 29.576 mL) with black caps.

    5. [ ] Under the fume hood, suction as much of the 50:50 liquid from the vial into the waste container as possible.
    ***Note: Avoid suctioning embryos into the waste container.
    6. [ ] Add 4% PFA fix to the glass vial so that it is ¾ full.
    ***Note: Wear gloves – fix is toxic.
    7. [ ] Suction the liquid from the bottom of the vial into the waste container.
    8. [ ] Repeat step 6.
    ***Note: Not necessary to add fix again if you are sure that you were able to take out all the liquid in the vial before adding the fix the first time.
    9. [ ] Place glass vial with embryos in 4% PFA fix for at least three hours at room temperature on the nutator or overnight in the fridge at 4°C.
    ***Note: Situate vial so that it is placed on it horizontal side on the nutator or in the fridge.

    Day 2:
    Goals: To prepare zebrafish for storage and slicing.
    1. [ ] [ ] [ ] Under the hood, remove fix and rinse embryos 3X for 5 minutes in PBS. Dispose of fix properly under the hood in the fix waste container.
    2. [ ] Add 30% sucrose in PBS to the glass vials containing the embryos and place on the nutator for an hour and 30 minutes or until embryos sink.
    ***Note: Use glass pipette to transfer embryos to glass vial because the embryos tend to stick to the plastic pipettes.
    3. [ ] Add OCT to mold, minimizing the bubbles.
    ***Note: Add enough OCT to submerge the embryos, exact quantity depend in personal preference.
    4. [ ] Transfer embryos by dipping embryos from sucrose in OCT and then transfer them to plastic molds with OCT (approximately four fish per mold) with heads facing the side with writing or labeled ‘H’. Gently swirl OCT with forceps until embryos are centered and oriented appropriately (embryos should be situated upright and heads of the embryos should point to the labeled side).

    ***Note: Absorb as much excess sucrose as possible with a Kimwipe, but do not touch the embryos.

    5. [ ] Place the blocks on the a large petri dish and seal the whole dish in plastic wrap and aluminum foil. Then, freeze in -80°C freezer for a minimum of 20 minutes.
    ***Note: Maximum time I kept the blocks in the freezer was 2 to 3 weeks.

     

    Day 3:
    Goals: (a) To slice zebrafish using the cryostat. (b) To permeabilize and block the zebrafish slices. (c) Introduce the primary antibody.
    Visual: Demonstrates the following steps regarding how to cryostat.
    http://www.youtube.com/watch?v=gIRW13XAHC0

    1. [ ] Set cryostat temperature to -21°C. Leave tools inside the cryostat so that everything maintains a constant and cold temperature.
    2. [ ] Place plastic molds in the cryostat from -80°C freezer and wait 10 minutes.
    ***Note: Number of plastic molds depends on the number of slides needed for experiment. One block may be used for two slides.

    3. [ ] Turn on the light in the cryostat chamber and take OCT blocks out of the molds, use a blade to cut off excess OCT (be sure that zebrafish are not embedded in the area).

    4. [ ] Put a layer of OCT on the chuck and immediately place a OCT block, positioned upright, on the chuck. Place chuck in the holder and wait until OCT on the chuck turns while which indicates that the OCT is frozen.
    5. [ ] Place the chuck with the OCT block into the clamp. Adjust the knobs to obtain an angle at which the OCT blocks are cut evenly.

    6. [ ] Change the cryostat blade if the cryostat has been left overnight because the freeze-thaw cycle can create buildup that will affect sections.
    7. [ ] Use a larger thickness (~40 microns/revolution) to make the first few slices into the OCT in order to get to the zebrafish. However, when sections intended to be put on a slide, the thickness should be set at 20 microns/revolution.
    ***Note: Use Superfrost slides only and place sections on the side with the frosted area because the sections will stick to this side due to charge difference.
    8. [ ] When finished, turn off the light in cryostat chamber.
    9. [ ] Reset the arm for the next user.
    10. [ ] Clean up the area. Scrape off as much OCT as possible from the chuck, thawing it outside of the cryostat afterwards to ensure that the next user will have a clean chuck.
    11. [ ] Thaw sections at room temperature for 10 minutes.
    12. [ ] Set up a slide box with wet kimwipes.

    13. [ ] Place 1 mL of 100 mM of glycine (1.86 g/250 mL in distilled water) on each slide for 1 to 2 hours to decrease autofluorescence.
    14. [ ] Remove the glycine by tipping the slide to one side and incubate the slides in PBS + 0.1% Triton (100 μl/99 mL of PBS) + 0.2 g BSA (PBT) for 1 hour and 30 minutes to permeablize tissue.
    Visual: Demonstration of how to remove solution from the slide
    http://www.youtube.com/watch?v=R3WgID1_op4
    15. [ ] [ ] [ ] Rinse 3X for 5 mins in PBT.
    16. [ ] Block by overlaying each slide with 1 mL of 5% goat serum (2.5 mL goat serum) + 0.2 g BSA per 50 mL PBS for 1 hour or longer at room temperature.
    ***Note: Other blocking serums, such as sheep, may be used instead of goat serum.
    17. [ ] Remove block and replace with primary antibody diluted in PBT.
    ***Note: Cover each slide with a piece of parafilm to prevent evaporation.
    Visual: One method of applying and removing parafilm
    http://www.youtube.com/watch?v=R3WgID1_op4

     

     

    18. [ ] Incubate for a few hours at room temperature or overnight at 4°C.
    ***Note: Overnight incubation with primary antibody recommended.
    Day 4:
    Goals: To rinse the primary antibody and apply the secondary antibody.
    1. [ ] [ ] [ ] Wash 3 times for 10 mins in PBT in the dark.
    **NOTE: Fluorophore-conjugated secondary antibodies will bleach if exposed to light. Always keep antibody stock and slides away from light to protect the fluorophore.
    2. [ ] Add secondary antibody (1:1000 Alexa 488 goat α mouse) diluted in PBS and incubate for 1 hour and 45 mins.
    3. [ ] Remove secondary antibody and overlay with Hoechst stain (1:1000) diluted in PBS for 5 mins.
    4. [ ] [ ] [ ] Wash 3 x 10 mins with PBT.
    5. [ ] Add mounting medium (Fluoro-Gel, with TES Buffer) and coverslip. Store at 4°C.

    Results: (Located: P:\Biology\Lom\STUDENT RESEARCH RESULTS\KHAN 14 (Slitrk5 Ab & AT zfish)\Spring 2013\Data)

    Antibody (what the antibody stain), Animal, Age, Brain Area
    Image

    Zn8 (stains cell adhesion molecule related to DM-Grasp), Zebrafish, 72 hpf, Retinal Ganglion Cell Layer (GCL)

    Anti-TH (stains for thyrosine hydroxylase), Zebrafish, 72 hpf, thalamus/hypothalamus


    SV-2 (stains for synaptic vesicles-2), Zebrafish, 72 hpf, Spinal Cord


    Elavl4 (stains for HuD protein), Zebrafish, 72 hpf, Tegmentum