This is a page of tips and suggestions for problems commonly encountered during frozen sections, taken from personal experience, with help from PAs, attendings, and online & book resources. It is not a comprehensive manual.
It helps to have some understanding about histotechniques. Observe and talk to your histotechnicians. :) http://library.med.utah.edu/WebPath/HISTHTML/HISTOTCH/HISTOTCH.html
Please edit this page or start a discussion if you have other suggestions, questions, or feedback!
Problems while trying to get a touch prep:
- "I put OCT on the tissue before doing the touch prep!" -- rinse it off with acetone/ethanol, wipe, let it dry, then do the touch prep or smear. Better than using water b/c it dries more quickly and causes less freezing artifact.
Problems while grossing a specimen for frozen section:
- "The ink is beading up and making a mess" -- wipe the specimen with acetone/ethanol before inking. If using q-tips for inking, dab it onto tissue or gauze to get rid of excess ink before using. You can also dip the inked specimen into dilute acetic acid and pat it dry; this will adhere the ink to the tissue.
Fatty Lymph Nodes at grossing:
-Attempt to remove as much fat from the outside of the node as possible using a scapel blade. It's best to use an autopsy blade since it is duller than the usual 10 blades. Use non-toothed forceps to anchor one edge and use a "scraping" motion with the autopsy blade to remove the remaining fat. Take your time and it will pay dividends at the cryostat when section and under the microscope for review.
Problems while freezing the tissue:
- "The tissue is cracking in my sections" -- put a warm finger on the block, then try again. The cracking is probably due to over-freezing. Warming the top gives it a smoother ice layer for you to cut. Also check the cryostat temperature.. keep it around -20C.
- "The chuck is stuck inside the cryostat"--it's probably because water got in and froze in there. Use 70%ethanol to try and melt it (it has a lower freezing temp).
Problems while cutting the tissue:
- "There's a chunk of OCT missing from the block and I can't catch the tissue" -- smear a little bit of OCT onto the block and put something flat on top of it (or put it face-down on something flat) within the cryostat. Re-trim and try again.
- "The tissue is on the edge of the block and rolls up"--try to give yourself two tissue-free corners opposite to each other so you can catch one corner and let the other corner stay stuck on the block as you pick up the tissue from the middle.
- "Section is stuck to brush and rips when I try to pull the brush away"--roll, instead of tugging, the brush away.
- "The tissue is too fatty and makes holes in my sections"-- I've seen a video of someone gouging the fat out and smearing OCT into the hole, but I've never done it b/c i'm afraid I'll gouge out something diagnostic... try cutting thicker sections, and putting a warm finger on the block then freezing with a blast of CO2.
Sectioning Fatty Tissue:
-Orient the specimen in the center of the chuck parallel to the blade. Upon sectioning, slowly go through the frozen permount until you reach the specimen and slight cut into it. Once you have a bit of a tag under your brush, coordinate the speed of advancing the wheel with your right hand to the guiding motion of the brush towards you. Try to cool the specimen as much as possible with the CO2 or dipping it liquid nitrogen. You can also add cotton gauze to a long cotton tipped swab and roll the tip into a point for finer placement. Dip the end in liquid nitrogen and apply the tip to the specimen.
Problems while getting the tissue onto the slide inside the cryostat:
- "Tissue doesn't stick to slide"--the temperature difference helps the tissue melt and stick to the glass. Don't leave the glass slide in the crystat. Breathe on the slide or touch it on the back to increase its temperature before lowering it into the cryostat.
Problems while staining:
- "The tissue keeps falling off in the staining solutions" -- couple of possibilities - it's cartilage, it's too thick, or you're using a non-charged slide. Suggestion for cartilage: pick up the solution containers or use a dropper, and stain the slide at an angle to prevent the tissue from running off the slide.
- "The colors don't look right"--Find out what kind of staining you use--progressive or regressive--so you can adjust your staining technique and/or figure out which solution is bad. Check the solution colors, and pH if you can/if applicable. Replace the solutions when you can.
Problems while coverslipping:
- "Bubbles!"-- May be due to inadequate dehydration (slide still has etOH) or mechanical entrapment. Dip in xylene with the coverslip still on. Press out the bubbles. If this fails, dip in xylene and carefully remove coverslip and re-try, with another dip in xylene and more glue.
-Prevent bubbles by placing the coverslip on several sheets of paper towels and laying a generous line of permount lengthwise close to the edge. With the glass slide, approach the coverslip at a 90 degree angle (perpendicular) to the coverslip. As the permount comes in contact to the glass slide from the coverslip, allow the permount to seal the entire edge. Lay the glass slide down slowly onto the coverslip allowing the permount to travel from the sealed edge to the open edge. It's usually better to have too much permount than too little since you can wipe the excess off.
- "There are clumps in the hematoxylin"--Some hematoxylin solutions need to be filtered before use. During the one day this happened to me, it didn't cause too much problem with the staining results but I had to do extra dips in the water to wash off the clumps and I guess theoretically the staining could become uneven. Also check to make sure tissue isn't falling off the slides and causing the clumps...