The cracks exist on the surface of paraffin blocks and due to this issue, all sections which I get are cut from those parts. Please guide me, thanks.
Hi Elham
it seems from your question that you have used manual embedding procedure. So, cracking may be due to the sudden cooling of the paraffin block while it was hot. So, if you have processed it manually, leave the paraffin block to cool at room temperature, then transfer it to 4 C0.
yes i am agree with Mr ramadan said; We have to put just a little among of paraffine before positionning organs cuts and cooling the blocks(to fix organs), then we completed with the rest of paraffin before it became cool, none you will have a biphasique blocks what will broke at microtome. secondly it may be due to the fact that the temperature of your refrigeration supply is not cool enough
This is all that I did.
Actually, instead of fresh tissues, I used frozen tissues that stored in -80ºC for long time (around 6 months). My tissue sample is the rat’s brain. After removing the brains from skull, I’ve stored them in -80ºC, without keeping in fixation reagent.
Recently, I took them out of -80ºC, put them in paraformaldehyde 10% (PFA) for 4-10 days. Then tissues were put in tissue processor for dehydration, in ethanol (50%, 70%, 80%, 95%, and absolute ethanol), and xylene , and then paraffin (totally 12 steps, 1 hour for each step) for overnight.
After that, I used manual embedding procedure to make tissue blocks. Left the blocks in cold plate of the embedding machine for a while, then kept them in freezer (-20ºC). After 1-3 days, I started to do the sectioning part by sharp blade of microtome. In this step, I faced lots of cracks on the surface of my blocks either on tissues or around them. I don’t know, is it because of the temperature only, or other problems refer the previous procedures.
I’m concern because I used the frozen, not fresh tissues. After some months used them and put in PFA.
Hi Elham
The procedure you are following is a standard one. the only problem i see is use of frozen tissue instead of fresh one. Also paraformaldehyde treatment was quite strong. usually 4% PFA for 1-2 h is sufficient.
Thanks dear Vijayendra. ooo. So I don't have any choices right now. Only have the frozen tissues. Do you think if I use the 4% PFA for 1-2 h the cracks problem on blocks will be finish or other things? This is very important for me. Thanks
I see that the problem may be due to the sudden cooling (from cooling spot to -20 c) of the paraffin block and/or inadequate clearing.
I am agree with Vijayendra Agrawal and Gaber Ramadan. Try with fresh samples and PFA fixation. This is the protocol I always use and works OK!
Hi Elham
Well, for now... there can be one thing worth try. Get your sample out of block by melting paraffin. Then embed again. this time after the paraffin solidifies on cold plate, transfer the blocks to 4 degree for a while. then try making sections.
But for better results, I would recommend to start with fresh samples, if possible.
Good luck!
Thanks all for your help. Unfortunately all of my samples are frozen. Do not have fresh samples and can not do them again. But I can try with my colleagues' fresh samples and see the results.
Sorry again, dose anyone have an experience of working with Timm Staining (Mossy fibers Staining)? I'm looking for complete protocol and all needed reagents for it. I searched through internet, they are different protocols and reagents with different percentages.
Have a look at this thread on RG:
https://www.researchgate.net/post/Can_someone_advise_on_Timm_staining_protocol
Hello Mam Elham. I just stumbled upon your concern which is dated approximately 4 years ago! By now, your studies may have been finished already, however allow me to share my inputs on your experience. Since you could not have obtained new specimens anymore , it is logical that the specimens be melted and re-embedded again in paraffin as suggested by some of your respondents. After which the paraffin is allowed to cool down under room temperature gradually . I believe that there is no reason to store the paraffin blocks at (-20ºC) as indicated in your protocol. PPFE blocks are NOT recommended to be stored at this temperature, but ambient, humid-controlled room temperature storage environment will suffice. Then, when your ready to section your paraffin blocks with the standard microtome, cool them liberally by immersion in iced-water or perhaps placing them quickly on the cold plate of the embedding center just prior to sectioning. I know my comments may come in a bit late at this time, but I do hope you may find this contributory and helpful for your future endeavors. With best regards- ROWEN T. YOLO, MD, Dept of Anatomic Pathology, University of Santo Tomas, Manila, Philippines. t
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