EFIC Sectioning Tips for Paraffin and PEG Embedded Samples
(Openlab 3.1.5 Software)
  1. Turn on lamp, filters, camera, and controller.
  2. Turn on computer (make sure this is done last for software to recognize that everything else has been turned on).
  3. Log in.
  4. Double click on OpenLab3.1.5.
  5. Go to Windows » Palettes » open aux filter wheel.
  6. Go to Windows » Palettes » open main filter wheel.
  7. Click level 3 for both filter wheels.
  8. Go to Windows » Palettes » shutters.
  9. Remove embedded sample from metal mold.
  10. Using a razor, trim the edges of the embedded sample.
  11. After trimming, place the embedded sample into the chuck of the EFIC system.
  12. Set the feed at 4um.
  13. Set the cut speed.
  14. Align the sample and chuck with the microtome blade, making certain not to trim the sample.
  15. Trim sample at 20-40 um feed until heart seen.
  16. Stop trimming, reset feed to 4um and speed to ~26.5.
  17. Use camera, shutters, and lens to center image on capturing screen (note: be careful not to bleach sample).
  18. Use navigation tools in OpenLab to adjust exposure, gain (brightness), and offset (blackness level) (Exposure levels will be higher for heart or heart/lung samples than complete embryo samples).
  19. Set magnification.
  20. Use coarse and fine adjustment knobs to focus image on desired magnification (2.0 or 2.5x for neonates).
  21. Set automation before heart area of sample is reached.
  22. In dialog boxes that appear, enter 0, then 1000, then G, then click "continue", then click "ok", (note: sectioning at 4um is optimal for reconstruction and resectioning).
  23. Watch sample meticulously, to make certain no wax sticks to sample if so, brush off gently. If wax sticks to the microtome blade, brush off blade very carefully; every so often, one can also clean blade with alcohol.
  24. After images are collected, go to file menu, select save as, save in appropriate folder as OpenLab LIFF format with the following template as filename: familyname_age_magnificationx_feed.