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    • CommentAuthorjuruble
    • CommentTimeFeb 1st 2008 edited
     

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

    Protocol revised: unknown

    Protocol written by: Barbara 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 four 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. Replace PBS with 30% sucrose in 0.1 M PO4 buffer solution until embryos sink (you can cut tails off tadpoles and pierce skin a few times for better saturation).
    4. Transfer embryos from sucrose to plastic molds 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).  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. 
    11. 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:

    1. Turn cryostat light on.
    2. Leave tools inside the cryostat so that everything maintains a constant and cold temperature.
    3. 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.
    4. 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 20 microns/revolution. 
    5. 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.
    6. 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).
    7. 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.
    8. 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.
    9. 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.
    10. 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.
    11. 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.
    12. 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.
    13. Label slides in pencil on the frosted area with the date, tadpole stage, group, etc.
    14. When finished, turn off the light in cryostat chamber.  Reset the arm for the next user.  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.
    15. Store slides in a slide box or folder at -20 degrees Celsius (freezer) until use (the sooner the better).
    • CommentAuthorjuruble
    • CommentTimeFeb 1st 2008
     

    You can also use the automatic feature to make sections. The buttons used to do so are locating alongside the armwheel lock button.

    Press the middle button once and the light on the left will appear, indicating that one section will be made when the green button is pushed.  Press the middle button again and the light on the right will appear, indicating that continuous sections will be made when the green button is pushed, stopping only when the green button is pushed again.  Push the button in the middle again to turn off this automatic feature.

    • CommentAuthorjuruble
    • CommentTimeFeb 1st 2008
     

    Chris Healey noticed that when students are wearing NYLON or other staticky clothing, the sections don't come off in a neat ribbon but are pulled every which way, presumably by the static.  So take note of what you're wearing before you begin!