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    • CommentAuthorkaswart
    • CommentTimeMay 13th 2010 edited
     

    This protocol explains how to: use the cryostat sectioning machine to obtain sections of a zebrafish embryo.

    Protocol written by: Kayla Swart (modified from cryostat sectioning protocol for Xenopus, written by Dr. Lom)

    WARNING: The cryostat blade is extremely sharp, so exercise caution when your fingers are near it. Push in the side bars above the blade when adjusting the chuck. Always clean the blade with brushes or swabs dipped in ethanol, not your fingers. Always label everything you prepare in the lab.

    Tissue Preparation:
    1.) Fix embryos in 4% paraformaldehyde for 3 hours at room temperature, or overnight at 4 degrees Celsius (fridge).
    2.) Rinse embryos in PBS three times (approximately 5 minutes per rinse). Leave embryos in PBS until you are ready to section them. Embryos can be stored in the fridge until sectioning, but keep the days between fixing, sectioning, and staining as short as possible for better sections and staining.)
    3.) Place fish in vials containing 30% sucrose and PBS until embryos sink (10 minutes to one hour).
    4.) Transfer embryos from sucrose to plastic molds (approximately 4 fish per mold) with heads facing the side with writing. Absorb as much excess sucrose as possible with a Kimwipe, being careful not to touch the embryos.
    5.) Add OCT to mold, minimizing the bubbles. Gently swirl OCT with forceps until embryos are centered and oriented appropriately. Freeze in -80 degree freezer for a minimum of 20 minutes.
    6.) Prepare chuck by covering end with OCT (no bubbles) and refreeze.
    7.) Score OCT on chuck head with a new razor and refreeze.
    8.) Mark orientation of embryos on OCT with a pencil.
    9.) Remove embryos/OCT from plastic mold and trim excess OCT. Refreeze for a few minutes.
    10.) Quickly add a few drops of OCT to chuck head and press tissue block into it (use cold forceps if possible -- avoid using warm fingers). 11.) Refreeze for a few minutes. Be generous with the amount used to seal the embryo to the chuck head. After mounting the block, squeeze some OCT around the intersection of the block and chuck head to seal any gaps around the edges. If while slicing the block is popping off, just use more sealant than before.
    12.) Gently trim OCT away from embryos keeping all surfaces straight. Final block face should have all sides equal or could be trapezoidal, and as small as possible.

    Cryostat Sectioning:
    Note: Superfrost slides are significantly more expensive than regular slides because they are specially coated to hold the sections more effectively, so make sure you have the proper slides and minimize slide waste.

    1.) Turn cryostat light on.
    2.) Leave tools inside the cryostat so that everything maintains a constant and cold temperature.
    3.) Make as few adjustments as possible to the cryostat.
    -Chamber (box) temperature should be set at -20 degrees Celsius. Section thickness is determined by the knob within the cryostat.
    - You can use a larger thickness (~60 microns/revolution) to make the first few slices into the OCT in order to get to the tadpoles.
    - However, when sections are made to be put on a slide, it should be set at 16-20 microns/revolution.
    4.) The plastic guide plate is controlled by the front bottom screw and can be one of the most important and subtle adjustments you make to obtain good sections.
    5.) The yellow button in the center locks the side armwheel. Make sure the side armwheel is LOCKED before doing anything inside the chamber (so you don't squish your fingers on the blade).
    6.) To insert the mold into the clamp, turn the knob on the side of the clamp clockwise. Orient chuck so that the longest edge of trapezoidal/rectangular tissue block is parallel to the blade.
    7.) Bring arm forward or backward using the control panel on your left until the face of the block is right in front of the blade. Use the fine or coarse adjustments. Make sure the plastic guide plate is DOWN; oterhwise you won't get clean sections.
    8.) Start rotating the armwheel to trin the block. It may be possible to adjust the section thickness to about 50 um or you can use the coarse feed adjustment.
    9.) Clean off the blade with upward strokes (never downward, which would cut the brush and dull the blade) using the paintbrush that is already inside the cryostat. The top edge of the guide plate may also be cleaned.
    10.) Continue sectioning; sections should come off in ribbons if all is going well, but sometimes you can get good individual sections even if you are not getting nice ribbons.
    11.) Pick up usable sections with a room temperature Superfrost slide. Make sure the mount/chuck head holder is under the blade because you will be able to pick up sections more easily. Brace edge of slide and touch lightly and quickly to blade to pick up sections. Section should be put on frosted side of slide. keep blade clean.
    12.) Label slides in pencil on the frosted area with the group, date, etc.
    13.) When finished, turn off the light in cryostat chamber.
    14.) Reset the arm for the next user.
    15.) 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.
    16.) Store slides in a slide box or folder at -20 degrees Celsius (freezer) until use (the sooner the better).

    Protocol written by : Maddy McCreery
    Further instructions for cryostat sectioning

    1. Place your tissue blocks inside the cryostat chamber, and allow them to thaw (~20 minutes) to the temperature within the chamber before sectioning.
    2. Turn cryostat light on.
    3. All tools should remain inside the chamber so that they are maintained at the same cool temperature. The cryostat should be set to ~-20 degrees Celsius.
    4. For safety, lock the armwheel by pressing the key button while preparing to section.
    5. Use a razor blade to cut off the end of the tissue block to get closer to the zebrafish. This will save you time when sectioning and will prevent you from dulling the cryostat blade.

     

         

    6. Using fresh OCT, mount the block vertically to a chuck by allowing the OCT to freeze within the chamber. Insert the chuck into the clamp by turning the clamp knob on the left clockwise. Position the chuck so that the longest edge of the tissue block is perpendicular to the blade.
    7. Unlock the armwheel. Using the control panel on the left of the chamber, adjust the chuck so there is an appropriate distance between the blade and the tissue block.
    8. Set the dial on the right of the clamp to ~50 um or use the coarse feed adjustment to trim down the block. Rotate the armwheel once for each slice until you have just reached the zebrafish.
    9. Reset the dial to 16-20 um to make thin sections for a slide. After setting the dial, rotate the armwheel one more time, as the first slice on a new setting will be the thickness of the last.
    10. Gently clean off the blade using upward strokes so as not to dull the blade.
    11. Label a Superfrost Plus slide using pencil, with your name, date, and zebrafish information. The slide should be at room temperature.
    12. Begin sectioning. To prevent sections from rolling, make sure that the anti-roll plate is down, or gently guide the section away from the blade with the end of a glass pipette or paintbrush. If you choose to use a tool, be careful not to pull as this could easily tear the tissue.
    13. Pick up each section by gently placing the room temperature slide on top of the section. Touch lightly and quickly so the tissue neatly adheres to the slide. Continue sectioning. Sections on the slide should be parallel and evenly spaced.
    14. When sectioning is complete, clean up the area. Pull off all OCT from the chuck, clean the blade, throw away any shavings in the chamber, return all tools to inside the chamber, and turn off the light. Turn down the cryostat temperature to -14 degrees Celsius.
    15. Store slides at -20 degrees Celsius until use.