Standard Operating Procedure Reichert-Jung Ultramicrotome

1. Standard Operating Procedure Reichert-Jung Ultramicrotome Guide to Ultramicrotomy Training Required

2. Magnification Coarse Advance Knob Focus Binoculars Light Buttons (2) On/Off Reset Auto Advance: Fine Adjust Presets Green button-motor start/stop Handwheel

3. Block Holder Block angle adjust Knife Holder with tightener Knife rotator Knife lock - front to back Knife - Side to Side Knife base position triangle

4. Trimming the block Use a single edged razor blade, to shave off thin pieces of excess resin. Shape the block so that you have 4, ~45 slopedsides. The blade will dull, so move to a sharp part every few cuts. A trapezoid or square are good shapes for the block face, remember to keep the top and bottom parallel to each other. Before Trimming After Trimming Sample Top View Angled Side View Side View

5. Water level knife Setting up the Microtome Press and hold the reset button of the microtome’s control box. It will beep when it is done resetting. Check the “Knife base position triangle” Turn the coarse advance knob until the bottom line of the triangle is barely visible. Place trimmed block into Block base, tighten side screw. Place block base in block holder on the arm of microtome, (long edge of the trapezoid down), tighten side screw. Place Knife in knife holder, tighten side screw. Fill knife boat with water. Water tension should pull the water’s edge up to the knife edge. The water level should then dip, away from the knife edge. Unlock the knife and push it towards the block, about 2mm apart. Using the knife side-to-side adjuster, align the block to the side of the knife for thick sections, at the first place where the edge is flat. Using the block angle adjust, roughly tilt the block so the the face is parallel with the back of the knife. Before After Block knife

6. Smoothing a rough faced block (if you do not see a reflection) The block face surface needs to be smooth and shiny to see the reflection. Move the block down below the knife edge (with handwheel), and move the block forward (with the coarse advance knob) until you see the block touch the flat face (back) of the knife. Move the block back slightly so that it no longer touches the knife (<10 turn of the coarse advance wheel). Set the auto advance to 1000 nm, and press the green button. This will start sectioning the block, 1.0m at a time. Press the green button again to stop when the face has been cut smooth. Or turn handwheel to manually smooth blockface.

7. Alignment of Block face to the Knife (1of 3) Look in the binoculars. Zoom out all the way, and focus on the knife edge. Rotate the block holder in the arm so that the face is level. Rotate the base of the knife holder so that the bottom edge of the sample is parallel to the knife edge. With your right hand, turn the handwheel to move your block up and down behind the knife edge. Look for a reflection of the knife edge on the block face. (If the face is rough, this will be hard to see.) The distance from the reflection to the knife edge is equal to the distance of the knife to the block face. If you see the reflection, skip the next slide. Note: if the control box starts beeping, make sure the dot next to the handwheel is within the wheel’s row of dots. If it is, press and hold the reset button. Reflection

8. Alignment of Block face to the Knife (2 of 3) Using the reflection, you can see the angle of the block face relative to the knife. Move the handwheel up and down and watch the reflection. If the block is angled, the reflection/knife distance will vary as the block moves up and down. In this example, the knife is farther away at the bottom of the block than it is at the top of the block. Turn the block angle adjust knob, so the reflection is a consistent distance when you move the block up and down.

9. Final Alignment of Block face to the Knife (3of 3) Zoom in all the way, and refocus on the knife edge. Move the block towards the knife, Check all angles between knife and block Repeat the previous steps if you see anything out of alignment. Move the block closer to the knife. Here is a block that is properly lined up and very close to the knife. The black reflection is barely visible, about the thickness of the red lines.

10. Thick sections Set the auto advance to 500 nm, and press the green button to begin cutting sections - If you are too far away from the block no sections will be cut Incomplete sections indicate that block/knife alignment may not be correct - Either re-align or continue cutting until an entire block face is sectioned. Prior to collecting sections clean boat with piece of tape, then cut desired sections. Fill the knife boat and adjust the reflection. Press start/stop, section until 4-5 nice sections float away on the water. Press start/stop button to stop sectioning. Put 2-3 drops of water on a gel-coated slide. Use a bamboo stick or “glass ball on a pipette” to pick up sections. Dip the sections into the water on the slide, rotate until they float off. Place the slide on the slide warmer at 60 until all the water evaporates. Stain sections then observe with a light microscope. Check the quality of your sections; complete sections with the target cells, even staining, dark enough stain to see clearly. Once satisfied with the sections either continue sectioning or move to a new part of the knife, realign and thin section (see next page). If not satisfied with your location in the tissue, continue sectioning. The knife is dull if the sections look wrinkled, torn or have gouges, move the block to a new part of the knife.

11. Thin Sections Once you have cut to your area of interest, and are cutting along your desired angle, it is time to cut ultrathin sections for the TEM. Depending on your tissue, you may be able to only cut as few as 3-4 sections on your cutting surface before you need to move the knife over to a fresh spot. Thin sections require a very sharp knife. Set the auto advance to 70nm, and press the reset button. To cut thin sections, move to a fresh spot on the knife. Approach the knife, taking extra care not to hit the knife edge with the block. If you do take a chunk while approaching, the knife will be too dull to cut thins, move farther over. Move the block as close to the knife as you possibly can. Zoomed in all the way, the reflection should look about the width of a piece of paper. Start the motor with the green button. Since these sections are about a 1/10th of before, the cuts will take 10 times more passes to start. Once the blade starts cutting, wait till it is about to make full face sections. (It will probably start off by shaving thin spaghetti-like pieces.) Stop the cutting, clean out the boat with tape, then resume cutting. Note the color of the sections and refer to the interference color guide on the wall. The ideal interference colors are light gold or silver. If the sections are blue/purple/green, and they are not making sections every pass, ensure that all the set screws are tight, if they are, the knife is most likely dull already. Try to get as many silver sections as you can, or until the boat is about 3/4th full.

12. Pick up Thin sections with a grid Use a hair-stick (or 2) to arrange the sections in the boat. If the sections are large (grid size) isolate them. If the sections are smaller, group them into clumps smaller than a grid. The goal is to get as many sections as possible onto each grid. Use self-closing forceps to scratch a fine line around a formvar coated grid to tear the excess formvar. Pick up the grid by the rim, and dip it into the boat away from any sections at a 45 angle. Move the grid so it is underneath your clump of sections, lift the grid up so that it catches an edge of the section then slowly lift it up out of the water at about 15. Use the hair stick to encourage the sections onto the grid Place the grid section-side up on a piece of filter paper or set the forceps and grid under a lamp to dry the water for a couple minutes, then place the grid in a gridbox. Do not forget to write down what sample is on the grid either in a notebook or on the grid box index card. One large section per grid A Ribbon of Serial Sections As many as possible