Hello, I am freezing "normal" bovine muscle (literally it is steak I got from the grocery store), and the pathologist is telling me that there is freezing artifact. Looking at an H&E, there is some spacing in-between muscle fibers but not within the fibers themselves. Can anyone tell me if this is normal for bovine muscle or did I not remove all the moisture before I froze? The fibers themselves do not look round, either. Basically, the pathologist is wanting to make sure I know how to freeze muscle before I handle a human sample. Thanks!
If you use for staining H&E, how about fixing 10% neutral buffered formalin and paraffin embedded section?
"I am freezing "normal" bovine muscle" has to be defined in terms of "freezing technique" YOU definitely use ( if there is done a "technique" that usually can be called scientific...) Freezing artifacts in biological material (usually "tissue") not only may be 'minute' but also are to be expected and appearing every time and their "size", nature ("morphology" ) depending on several physical parameters [e. g. size of specimen, precooling, 'coolant', freezing rate, ...] . So - in order to give (a) serious answer(s) - the description of your "freezing technique" is an essential part of the solution to your question (which confirms "the pathologist is wanting to make sure I know how to freeze muscle before I handle a human sample" ). There is a lot of specific literature on "how to correctly freeze and handle" biological tissue - especially human samples for diagnostic purposes & for further examination, being it histological, immunohistochemical, immunocytochemical or other analytical techniques. Best wishes and good luck. NB/PS: even if you have frozen muscle from "bovine steak" PERFECTLY (in the eyes of the pathologist) it might be a quite different scenario to cryofix/freeze a human muscular specimen you'll get handed over from the surgical theater....
Thank you for the responses. I am using the isopentane/liquid nitrogen freezing method which I have been doing for years (I trained at Duke University). I don't typically have freezing artifact issues; however, I have never frozen post-mortem tissue - I've only dealt with surgical tissue. I think it's a good idea for me to point out to my researcher that the surgical specimen WILL appear different than freezing a steak! I'm attaching 2 pictures - please forgive the coloring. I'm having issues with the camera software and don't know how to get the picture to look as pink as the stain (this is just an H&E). The first picture is of the freezing artifact, i.e.: spacing in-between the muscle fibers. The second picture is of the same muscle that I thawed out, blotted the moisture off (more diligently this time), and refroze (please forgive the knife mark, I was in a hurry!).
I'd love to know all of your thoughts. Thanks!
Dear Adrienne, thank you for clarification regarding your skills, especially knowledge of the methods used for the freezing of specimens. Now I understand....
I also think (and therefore second your opinion) that "freezing a post mortem" steak tissue for sure might display additional artifacts which - taken the correct isopentane / LN2-freezing for granted - aren't joined to / caused by the freezing technique itself. Thank you also for providing the images (re colors: if you find elsewhere in your software a function 'autocorrection of white balance' just place an emtpy area of your slide in the light beam and push....) the "freezing artifact" you show in image 1 to me seems very common too also in (human) surgical specs. As one can see from the mag-bar: that spacing-in between the muscle fibers [=intrafascicular] happens to be located more in the middle of your spec (some 400-500 µm from the surface....) Also the interfascicular space seems to be dilated a bit (but we can't say something about collagen fibril network located there unless you will stain for collagen....) . Image 2 is quite fine (I only can see ONE intrafibrillary vacuole filled with inflammatory?/phagocytic? cells [= left margin, @ 10.00 ] and only four fibres (one right in the middle of theimage) which seems to display some disturbance (brighter, more lucent appearing roundish areas ) which might be interpreted as mild "freezing artifacts" (mild, because more severe artifacts consist of empty, lucent vacuoles, sometimes "filling" the whole fiber diameter). It is interesting that you get such an excellent morphology after a cyycle: freezing - thawing [cf: "'+blotted the moisture off (more diligently this time), and refroze" ] -freezing.
NB: more/less dilated interfibrilla r/ inter- & intrafascicular spacing-in can have other reasons than only handling the biopsy / specs prior to and / or freezing them for diagnostic purposes....
Best wishes and regards, WM.
I would NOT use steak from the grocery store as a control. The "meat" will likely be many days post mortem which means lots of autolysis artifact. Its also very common to at least partially freeze meat when butchered. That meat would not have been cryoprotected. I would go to your rodent facility where they are likely putting down hundreds of mice or rats a day and get some FRESH muscle. This should be flash frozen in 2 methylbutane or isopentane with dry ice to start.
Can you stain for SDH or succinic dehydrogenase after freezing to see whether basic enzyme can be viewed, then you can do a methylene blue alone serial to check if sample contain basic fiber types; Good luck, the tissue seems ok to do these techniques.
Dear @Adrienne: So - at least for me - there arises the question: what is the real 'rationale' for your freezing / (and thawing and freezing again, cryosectioning and staining "normal bovine muscle meat = steak" in comparison to human diagnostic tissue material you have to hand over to a pathologist for evaluation? Regards, WM
Ah....now that I read your answer # 04 I see the hint "however, I have never frozen post-mortem [NB: 'p.m.'] tissue - I've only dealt with surgical tissue." So can we / I assume you're in charge of or confronted with freezing p.m. muscular tissue [as collected from dead bodies / corpses from the morgue/autopsy theater as in Europe usually affiliated with a Pathology Institute] for research purposes.... I have done a lot of ultrastructural studies for diagnostic purposes (by using common and sophisticated fixation and processing techniques) from (living!) patients as well as dead bodies and therefore can tell that (at least after standardized chemical fixation and processing) muscular 'autopsy' material within 2 hours of patients' passing away shows severe alterations (including [ not only! ] vacuolization and generation of interstitial / interfascicular edema). My best regards to your "pathologist"..... and my compliments, WM
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