Microtome sectioning

Setup

Optional: Put block on microtome and cut layers of wax off until tissue starts being cut. Soak parafin blocks in ice bucket with ice water and a dollop of hand soap for at least 30 minutes prior to cutting.
Turn on water bath & fill with water, set temperature at 37°C.
Trim wax off the sides and back of blocks so that they will mount on the microtome in the same manner even after you take them off.

Preparing the machine (Leica RM2335)

Place paraffin sample in block holder (always in the same direction) using lever (M). I generally have the block's front end pointing to the right.
Set section thickness (normally 5 µm) using knob (F)
Set angle of blade (normally 3-4°) using allen wrench on right side of blade holder (N, can't see in this picture)
Insert blade in blade holder and tighten with lever (I).
Adjust the blade holder's lateral position by sliding the block holder back and forth after loosening lever (H). Can use different parts of the blade until all sections of the blade have been dulled by cutting. Generally replace blade if it has been used on more than 10 blocks or if it seems dull or creates tears in the sections.
Clean blade by using paint brush in an upwards (never downwards) motion.
Move sample (M) backwards (away from blade), using circular handle on left side of machine (C). Then use the same circular handle (C) to bring the sample towards the blade so that the sample is almost touching the blade.
Unlock the cutting wheel using lever (B) and flip the blade guard (J) down. Move the sample up and down using the circular handle on right side of machine (A) and watch from the side to see if the sample is parallel to the line of the blade. If it is angled forwards or backwards, or side to side and not parallel, adjust the tilt forward/backward using knob (D) or side/side using knob (E). Remember, that lever (G) must be loosened in order to make these adjustments. And always tighten lever (G) after making the adjustments.
Sample is now ready to cut.

NOTE: Check to make sure all levers are tightened at this point (G, H, I & K)

Cutting sections and transferring to glass microscope slides

Rotate right side handle until blade begins to cut sample. Note: Can rotate handle clockwise or counterclockwise, either way the sample will move towards the blade. To move the sample backwards you need to use the circular handle on the left side of the machine.
Optional: Set the section thickness to 25 ��m while you are initially cutting in order to cut through the block quickly to the desired location. Then re-set the section thickness to 5 ��m and cut once more to get the block to the position for collecting samples.
Cut the block and gently lift the section away from the black face plate using a paintbrush or your fingers, but do not pull on the section. Cut another section, and the second section will remain attached to the first section. Repeat this for as many sections as you want in a single ribbon of wax (generally two or three). Use a second paintbrush to lift up the ribbon of wax by pulling it off the blade. Gently transfer the ribbon of wax to the 37�� water bath using the paint brushes and lay it gently on the water.
Let samples float in waterbath for about 2 to 5 minutes. Place glass microscope slide in water and gently allow wax slice to adhere while lifting out of waterbath at an angle.
Label the glass microscope slide with 1) animal #, 2) tissue, 3) slice #, 4) date, and 5) your initials.
Place the microscope slide in a metal rack. When all slides have been inserted in metal rack, place the metal rack in a 60�� C oven and incubate overnight to melt the wax.
The next day remove the slides from the oven and transfer to slide boxes. Label slide boxes with animal #.

Reagents

Fisher microscope slides: Superfrost/plus cat# 12-550-15