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Mohs Histology Consulting Services

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Mohs Technical Tips to assist and expain useful technics to obtaining better sections

Tip #1. Liquid Nitrogen is much colder than any canned cryo-spray. With proper application to the fatty area in a specimen(after facing the block), one can obtain nearly perfect sections of fat. It ususally takes repeated applications of liquid nitrogen to achieve this so don't get discouraged if the sections don't get better right away. I prefer to use a liquid nitrogen sprayer (used for freezing warts and the like) and I hold the nozzle very close to the block face. This reduces overfreezing of the epithelium and minimizes blowing shavings around in the cryostat. Alternately you can use a large swab (like a vaginal swab) dipped in liquid nitrogen and held on the face of the block. Remember to discard the first section after freezing, as this one will be thin and unsatisfactory due to shrinkage anyway. Also, often it will be necessary to increase section thickness to 8 to 10 microns. When the fat is frozen sufficiently, it will cut well and appear as a glassy, translucent area within the section. Sometimes cutting the section faster(sometimes very fast) than usual will also help get the section you want. Good Luck!

Tip #2a Cutting Fat when you don't have liquid nitrogen is more difficult but still possible. It requires cutting sections much thicker than with liquid nitrogen, up to 14 or even up to 18 microns thick. It also requires the use of cryo-spray to cool the specimen down. Usually placing the embedded block face down on the cryostats cooling/embedding bar for 10-30 minutes with help a lot also. What I have found to be the biggest help when the block is cool enough is to use an anti-roll plate and cut the section very fast. Sometimes it is helpful to change knives when the fat won't cut. So you can cut thinner sections and then cut a couple of thicker sections this way to get perfect sections of fat. Again, remember to discard the first section after spraying with cryo-spray, as this section will be thinner due to shrinkage.

Tip #2. Cutting cartilage is not difficult as mohs technicians know ( it may be hard to get the specimen to lay down with cartilage, but it cuts relatively easitly) But keeping sections of cartilage from falling off the slide during staining can be a problem. Now that + (plus) slides are available, cartelage does not fall off quite as readily, but can still be a problem. Two things you can do to help cartilage stay on the slide (even regular superfrost slides) is to heat fix the specimen using a hair dryer or a 60 degree oven. Be careful not to use too much heat. Also, fixing the slide first in Acetone for one minute, before putting the slide in alcohol (or other fixative) will help.

Tip #3. The anti-roll plate. Many techs, expecially if they have been cutting Mohs frozen sections for many years, find anti-roll plates to be a nuisance and have developed a high degree of proficiency using a brush. Many older style plates were made of plastic and the adjustment required a lot of fiddling. The new glass anit-roll plates, especially those on the Leica (or Reichert-Jung) cryostats are designed much better. On the Leica 1800, 1850, 1510 and 1510-S cryostats, the anti-roll plate now comes with a removable glass plate, which effectively gives 4 replaceable edges to use before a new plate must purchased. Also, with this style anti-roll plate, the plate holder itself is designed to rest on the ends of the disposible knife to provide the gap necessary for the sections to slide underneath the glass.

There is one critical adjustment of the anti-roll plate which cannot be overlooked. The plate edge must be parallel to the knife edge. This is critical or the section will bunch up on one side or the other, even when the plate is adjusted front-to-back optimally. This is where many frustrations come from. Sometimes it is easier to visualize that the edge is parallel when there is no knife in the blade holder (for disposible blade holders). If the plate is not parallel, it can be adjusted by loosening the allen screw slightly and then adjusting the edge slightly. Note: With the new style plates, make sure the plate is all the way back in the holder and firmly secure with the two black screws which hold it in the holder(Leica cryostats).

When using the above configured anti-roll plates AND using a disposible blade holder (high or low profile) on the Leica and Reichert-Jung cryostats, it is important to center the knife in the holder. The metal fingers of the glass plate holder rest on the ends of the knife to provide the clearance gap. When you need to move the knife because of a nick or dullness, use the lateral knife adjustment (lever on the left of the knife holder on Leica cryostats) to move the knife over. For Microm cryostats, the glass plate has two usable edges and the grey strip must rest directly on the knife edge.Note: On older style knife holders for the Leica or Reichert-Jung 1800, the amount of lateral adjustment of the knife is limited, in order to still have the anti-roll plate fingers continue to rest on the knife because lateral knife adjustment is independent of the anti-roll plate.

Also, set the anti-roll plate down gently on the knife. Slamming the glass down will cause it to chip and may break the glass if put down hard enough. Also, let the anti-roll plate rest with its own weight on the knife. Don't push it down with the finger on the handle as this will cause the gap to be smaller and the sections to wrinkle up.

With a properly adjusted anit-roll plate you should expect the section to slide smoothly under the plate and be very flat. If it isn't, something is out of adjustment.

Tip #4 Brushes Brushes have been and are an important tool for the Mohs histotechnologist. Before the advent of quality, functional anti-roll plates, brushes were and are used by many techs to guide the section onto the knife during sectioning. In addition, use of brushes is important to manipulating the section when cut, to position it for easy pick-up and to flatten the section to remove wrinkles and folds. I have found the most useful brush for flattening out the section and brushing out small folds and curles in the epithelium(flips) is a #3 or #4 brown camel hair brush. Brown camel hair is soft and flexible and is more gentle on the tissue section. Red sable or synthetic, which is easier to find and less expensive, is much too stiff in my experience and is likely to tear the section. In addition, by trimming the camel hair brush at a 45 degree angle, one has a tool which is very useful for brushing out flips in the epithelium when used with the bevel side down and brushing gently, but repeatedly across the epithelial edge toward the edge of the OCT. When brushing out flips, hold the brush as a shallow angle relative to the section and brush from the epithelium outward (away from the center of the section).

Tip #5 Slides Slides are an integral part of producing quality Mohs sections. There are two main types of slides used today. Regular uncoated Superfrost slides and coated Superfrost "+" slides. The latter come in many configurations including HistoBond, etc, but they all have a coating on the slide to produce a positive charge. The purpose of this coating and the positive charge is to make sections adhear more tightly to the glass. This is most important when cutting sections containing cartilage. Cartilage is notorious for lifting off the glass during staining. Technicians usually heat the slides to fix the cartilage to the glass more strongly. Even with heat, cartilage tends to fall off on regular glass slides. One additional technique which will help cartilage adhear to the glass is to fix the slide initially in Acetone and then to air dry the slide before starting the slide on the stainer in the fixative of choice.

Superfrost "+" or Histobond slides are more than twice as expensive as regular Superfrost slides making their use for all sections unnecessairly expensive. For the vast majority of sections, uncoated glass slides will work very well. Note: If sections without cartilage are falling off the slide, it is usually because the tissue portion of the section is sitting on top of the OCT of the previous section. It is then not really on the glass and when the section is rinsed in water to disolve the OCT, the section starts to fall off. This problem can be fixed by trimming the OCT away from the sides of the tissue (leaving a small margin at the sides) before sectioning or by wiping the OCT off the slide before the next section is picked up. This allows the tissue of the next section to sit directly on the glass. It takes a little practice to wipe the OCT off cleanly, but once mastered, it becomes an automatic process done after each section is picked up.

When embedding using the slide method the least expensive, plain, unfrosted slides may be used. When using these budget priced slides, cleaning or reuse is not necessary. Many practices already have these slides on hand for performing KOH preparations.