Sectioning Paraffin Blocks

Transcription

1 Description Laboratory Standard Operating Procedure for: Sectioning Paraffin Blocks This SOP requires a Designated Trainer! This SOP describes how to section tissue embedded in paraffin wax with a Leica RM2255 microtome. Please refer to the microtome instruction manual for more detailed information. Potential Hazards Microtome blades are very sharp! They can cause a cut simply by handling the knife and can cause significant damage to fingers that are caught between the sample and the blade while sectioning. Paraffin left on the floor can cause the floor to be very slippery. Work Practice Controls Always keep the knife guard up and the hand wheel braked or locked when not actively using the microtome. Always brush/wipe up and away from the blade when removing excess paraffin. When clamping the various levers please secure them firmly but do not overtighten them! Personal Protective Equipment (PPE) Standard lab PPE. Transportation and Storage Water baths must be stored clean, dry and unplugged. Waste Disposal Unwanted paraffin sections or trimmings may be disposed into a regular trash bin. Procedure Start-up: Refer to the Control Panel diagram on page 22 of the manual. 1. Set a water bath to C. 2. Create an ice bath using crushed ice and deionized water. This bath should be mostly wet ice as the block face will need to chill and the tissue hydrate at the same time. 3. Turn microtome on via the toggle switch on the back of the machine; there will be a beep and the machine will initialize. The display should illuminate on both the machine and the control panel. Choose CONT (Continuous) Cut Mode. 4. Be sure that the chuck assembly is in the rear end position by pushing the Coarse Feed Backwards button. 5. Secure a low profile disposable knife in the knife holder using the clamping lever. Please start at the left side of the knife and move it to the left when a sharp edge is needed. If desired, the oil can be removed from a new blade (see Trouble Shooting Section, item A). 6. Brake or lock the hand wheel. The brake is the lever on the lower right side of the machine and the lock is at the top of the hand wheel. 1 Rev A: cw

2 7. Trim at least 1 corner from a block with a razor blade. This will show the orientation of the ribbon on the water bath and make sections easier to pick up. 8. Insert the block into the universal cassette clamp by pulling the metal handle towards you, which will open the clamp. Release the handle to secure the block. A block may be inserted horizontally or vertically. 9. Zero the position of the chuck by releasing the clamping lever on the chuck base and twisting both the horizontal and vertical alignment knurls until the leveling indicators are fully visible. The white line on the knurls should line up with the arrows. Sectioning 10. Select the Trimming Mode button and use the +/- buttons to set the trimming thickness in microns. 11. Release the clamping mechanism of the Knife Holder Base and move the base forward or backward to position the knife near, but in front of, the block face. Secure the clamping mechanism. 12. While looking at the block from above, use the Slow Coarse Feed Forward button (button with single arrow) to move the block close enough to the knife to cut a section. Rotate the hand wheel slowly to see how close the block is to the knife. If the block is stopped by the knife you have gone too far and must use the Backward button to make the necessary adjustment. 13. Rotate the hand wheel clockwise to take sections from the block until the tissue of interest (TOI) has been exposed; this is called facing the block. 14. Switch to Cut Mode using the TRIM/SECT button and set the cutting thickness. 15. Rotate the hand wheel a few more time to smooth out the block face, and then lock it. 16. Remove the block from the chuck and chill/hydrate it in the ice bath. Note: Keep pouring off water as the ice melts as you want an ice bath, not a water bath with ice. The amount of time in the ice bath depends on the size of the block, the type of tissue being cut and how dry that tissue is. If the block isn t cold enough the paraffin will compress too much during sectioning. If the block has been left on the ice bath too long the tissue will swell. Start with a few minutes and go from there. 17. Remove the block from the ice bath and quickly dab the face on a paper towel to dry it. 18. Be sure that the block and the knife are aligned correctly. If other blocks have been trimmed, the block face may be too far forward so proceed slowly until you know that you will not chunk the block against the knife. 19. Section a ribbon of paraffin slices at a steady pace using a brush and forceps to gently lift, guide and pull the ribbon. See Trouble Shooting comments below. 20. Take the ribbon, or a piece of it, and lay it on the water bath using forceps and a brush (or whatever works for you). The ribbon should flatten out immediately. The tissue may need a little longer to flatten but it may never be completely flat depending on the processing that was done. Note: NO razor blades are to be used on the microtome itself. A long ribbon can be cut into shorter ribbons by placing it on a clean bench top and using a razor blade. 21. Orient a charged slide next to the TOI by slipping the full length of the clear glass into the water at a angle next to the TOI. Move the slide underneath the TOI and touch it to the ribbon. Be sure that the start of the section is sticking to the slide. Lift the slide out of the water using the same angle, cutting the ribbon if needed. See Trouble Shooting comments below. Sectioning Paraffin Blocks 2 Rev A

3 22. Place the slide on a heated slide warmer (approximately C) to further flatten and dry. If immunostaining is to be performed, warm slides below 37 C or place them directly into a slide box. 23. Repeat, if needed, by chilling the block again. Cleaning Procedures 1. Turn off the microscope and the microtome. 2. Lock the hand wheel. 3. Carefully remove the blade with your fingers or use the magnetized end of the Leica brush. Used blades can be disposed in a sharps container or in the bottom of the blade box. Note: a knife that still has a lot of clean area can be left in the machine if the knife guard is up and a large note is left in the cleaned paraffin waste tray. 4. Brush loose paraffin from the machine into the waste paraffin tray. Empty the paraffin tray into the trash by pulling up and out from the machine until the magnets release the tray. Insert the empty paraffin tray in the reverse manner. 5. Wipe all stuck paraffin from the machine including all around the handle (gauze works great for this). 6. Wipe stuck paraffin from the tools you used, the chair and the floor. Leaving paraffin on the floor makes the floor very slippery! 7. Empty the ice bath in the sink and leave it face down on the drain board or peg board. 8. Turn off, unplug and empty the water bath in the sink. Wipe any stuck paraffin with gauze while the bath is still warm as this makes cleaning easier. Use a paper towel to dry a metal water bath. A glass insert can be left to dry in the drain board. 9. If a knife has been left in the machine, be sure that the knife guard is up and that a large note has been left in the paraffin tray saying Caution, Sharp Blade! 10. Cover the microtome and the microscope. 11. Record your use in the Microtome User Log book. Trouble Shooting A. Sections that curl There are several reasons why the first section will curl and separate itself so that a ribbon can t be formed. a. New blades have a thin coating of oil on them from the manufacturing process. Remove most of this oil by lightly pinching a piece of gauze around it from the bottom and swiping across the entire blade. Turn the blade around and repeat from the other side. b. The blade is actually too sharp and needs to be conditioned. If a new (or new part of a) blade is to be used, try to use it first by doing the final trimming of a block in this area. When moving the blade to clean areas leave some of the used area, too, to prevent curling. c. At the start of a curling section, place the small brush above the curled beginning and lightly hold it there. Move the brush with the paraffin while taking the rest of the section. Your next section should form a ribbon. If not, repeat the action until it does. Sectioning Paraffin Blocks 3 Rev A

4 B. Cutting a ribbon on the water bath Because at least 1 corner of the block has been trimmed, separating a portion of the ribbon shouldn t be too difficult if sections are taken perpendicular to the ribbon. Taking sections horizontal to the ribbon can be more difficult. a. Sometimes just moving the slide back and forth at the junction between 2 sections will cause the ribbon to separate. b. Use the corner of a slide to perform multiple quick chops through the ribbon so the sections will be easier to separate. C. A ribbon is difficult to produce: a. Thick/thin or chattering If the sections in a ribbon are obviously of differing thickness, re-tighten all clamping levers. Also check that the block of paraffin is in complete contact with the cassette. Air holes between the two can cause the block to be unstable. Be sure the block has been trimmed of excess paraffin all around the edges. b. Chill the block longer. c. Use the shortest time possible when taking the block from the ice bath to sectioning. d. Increase or decrease the thickness of the sections. Injuries Immediately report any knife cut to the Histologist. If the employee is in need of emergency medical attention, call 911 immediately. TREATMENT FACILITIES: U-M Occupational Health Services -- Campus Employees Mon-Fri 7:30 am - 4:30 pm After hours - go to UM Hospital Emergency Dept. Urgent Care Clinic C380 Med Inn building 1500 East Medical Center Drive, Ann Arbor (734) University Health Services -- University students (non-life threatening conditions) Mon-Fri 8 am 4:30 pm, Sat 9 am 12 pm Contact for current hours as they may vary 207 Fletcher Street, Ann Arbor (734) UMHS Emergency Department -- after clinic hours or on weekends 1500 East Medical Center Drive, Ann Arbor, (734) Report all emergencies, suspicious activity, injuries, spills, and fires to UMPD by calling 911 or texting Training of personnel All personnel are required to complete the Comprehensive Laboratory Safety session (BLS042w or equivalent) via MyLINC. Furthermore, all personnel shall read and fully adhere to this SOP when performing this procedure. Certification I have read and understand the above SOP. I agree to contact my Supervisor or Lab Manager if I plan to modify this procedure. Sectioning Paraffin Blocks 4 Rev A

5 Name Signature Trainer s signature Date Principal Investigator / Safety Officer Date (signature) Printed name: Carol Whitinger Designated Trainer Printed name: Carol Whitinger (signature) Date Sectioning Paraffin Blocks 5 Rev A