I have frozen rat kidney and I want to do a histological or pathological study. Can I use frozen tissue or I have to defrost them? If I defrost them, will the integrity of the tissue be destroyed after defrosting or not? Thanks for your attention
Basically it is no problem to transfer the sample into buffered formalin and then process the tissue for paraffin embedding. However, the morphology of the tissue is compromised by freeze artefacts.
Hi,
What you are looking for is cryosectioning.
Check it on google.
Yea defreezing will cause tissue destruction in most parts and you wont get good/real results. So try cryosectioning but its not 100% that it will work properly also...
Of course, fixing fresh tissue in 10% formalin will give you the best slides for histopathological examination. However, in the past we have fixed brain and liver tissue that had been stored at -70oC and the integrity of the tissue after fixation was very good.
Take ice cold 10% formalin (keep it on ice) and put your tissue straight from the freezer into the ice cold 10% formalin. Leave it in the fridge for 24 hours (depending on size of course) and than you an store it at room temperature. Good luck.
If the tissue was properly frozen (snap freezing) and conserved in the freezer, may be better to get sections with cryostat of thickness from 5 to 10 - 15 um depending on the use (morphology alone or histoenzymatic stains) and the skills of the operator (OCT embedding may be not necessary, but only a small quantity to bind the sample to the cryostate chuck).
In alternative I use to drop the frozen sample directly in the fixative (I avoid formalin for safety problems) without thawing before fixation. Some artifacts will be present but possibly morphology is not terribly affected.
Hello
One time I tried frozen tissue, I defrosted it on white ice and fixed in 10% formalin. Then processed to prepare paraffin section, the section was good and the staining was great. You can try one or two samples.
Thanks
Cryostat used for staining lipids in cells as well as enzymes
The structure disintegration happens when the ice melts at about zero degrees, yet many chemical activities can occur below freezing temperatures. I would try immersing some of the tissue sample in fixative at -20 degrees (or maybe minus 10, usual frig freezer temp) then go through the normal wash and dehydration series in the freezer for embedment in Lowicryl. This resin is polymerized in the freezer with ultraviolet light. Then once embedded you can do all kinds of wonderful things like immunogold and TEM.
See J Histochem Cytochem. 1984 Nov;32(11):1217-23. Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. Altman LG, Schneider BG, Papermaster DS
The abstract supplies adequate instructions. http://www.ncbi.nlm.nih.gov/pubmed/6436366
Basically it is no problem to transfer the sample into buffered formalin and then process the tissue for paraffin embedding. However, the morphology of the tissue is compromised by freeze artefacts.
In my experience quality of sections after fixation (buffered formalin or suitable formaldehyde-free fixatives) depends on accuracy of the freezing process. Snap freezing with controlled low-temperature - as used for muscle biopsy - may give good results. Long time storage at low temperature may also dehydrate tissue if not kept in air-tight envelope.
Yes , but in this case (Freezing microtomy or cryotomy ) ----directly after cutting the specimens we make staining according the purpose , for example if we are study the fats -----use Scarlet red or oil red o or acridine ornage or osmic acid ---etc
Sure, but you should use it directly without defrost. Just sectioning using cryostat and staining sections directly accordingly your methodology,
Yes , you can use cryostat microtomy to cut the tissue sections. After mounting on glass slides, please cool down to room temp infront of a slow running fan. After that you have to fix with alcohol and continue any stains such as H&E or IHC.
Yes I used the Crostat method for cases I was treating , Norman Thomas PhD FRCD Chancellor ICCMO Prof Emeritus U.of Alberta
Oh nice! Thank you for your valuable answer sir! This would a be great help for a student who is planning to do a class project on EM. Regards
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