Sectioning using the LKB 8800A microtome:

1. Using a new blade make a pyramid on the tip of a block with the specimen at the tip of the pyramid, if necessary shave the tip to expose the specimen. Make sure the tip of this pyramid is less than 0.5 mm on each side.

2. Take fresh grids of type G300-Cu and place them in a small vial with acetone.

3. To clean the diamond knife, first take a Q-tip, dip it in alcohol and use it to clean a new metal blade. Use this clean new blade to cut a 45o wedge from a styrofoam stick. Dip the freshly cut styrofoam wedge into clean 100% ethanol and pass it along the diamond knife blade.4. Mount your sample on the microtome.

5. Fix the cleaned diamond knife on the microtome.

6. Place wet paper towels around the area of diamond knife to reduce static in the cutting region.

7. Align the pyramid relative to the diamond knife. There are four alignment steps. (i) Looking from the side of the microtome, the pyramid has to be at the same height as the blade when the arm is locked. (ii) Looking from the front through the binoculars, the top and bottom sides of your pyramid have to be parallel to the edge of the blade. (iii) Looking from the top through the binoculars, the face of the pyramid has to be parallel to the blade. (iv) Looking from the top through the binoculars, the pyramid has to be brought as close as possible to the blade without touching the blade (turn the large macrofeed knob clockwise at first and then turn the small macrofeed knob).

8. Add distilled water to your knife boat. If you are using the Microstar standard boat, use 600ul of water. Use the side of a freshly sharpened orange wood stick to wet the edge of the boat and bring the water close to the blade with the tip of the stick, DO NOT TOUCH THE BLADE WITH THE STICK. If water does not stay near the blade, rub the inside of the blade with wet tip of the stick. It is not necessary for the water to be at the edge of the blade at this point, when the first complete section is cut, the water will be drawn to the blade.

9. Release the arm of the microtome, set the speed to 5 m/sec, and set the control knob to auto. If the arm does not swing up and down, changing from manual to auto a couple of times.

10. Turn the small macrofeed knob clockwise one click at the time until you get your first section. Each click is one micrometer and so the first section may be as thick as one micrometer.

11. Once you get your first section, change the cutting speed to 0.5 m/sec.

12. Turn ON the FEED and set it to 600A or 700A (this is not the actual thickness of your section).

13. The microtome can now be left to section on its own for about half an hour, you may need to adjust the feed control to increase or reduce the thickness of your sections. Remember, purple is too thick and silver is too thin, gold sections are ideal for our TEM.

WARNING: Do not leave the microtome unattended for more than 5 minutes.

14. Once you have obtained enough sections, retract the knife, lock the arm, and turn off the feed.

15. Dry your grids, which have been soaked in acetone for more than 20 min by now. Swirl the vial and pour the suspended grids on a piece of lens paper.16. Group the sections using a moist eyelash on a wooden stick.

17. Collect approx 7 sections on the shiny side of each grid by simply touching the sections floating on the boat with the shiny face of the grid. Collect only the gold sections.

18. Place your grid with sections on a lens paper, the shiny side of your grid which contain your sections should now be facing up to prevent the paper fibers from touching the sections. Let your sections dry for 5 min on the lens paper and either store them on a grid box or proceed with the staining.

19. While the sections are drying, clean the diamond knife by rinsing it with distilled water, followed by dust off and a freshly cut styrofoam wedge dipped in ethanol. Cleaning the diamond knife immediately after sectioning is extremely important. Put the cleaned knife on its storage box.

Dont forget to clean the area, put all the tools away, and cover the microtome.Electron Microscopy Center (212) 650-8591

The City College of New York

Please send comments and corrections to morales@sci.ccny.cuny.edu