I want to perform colocalization of two markers (stem cell marker & angiogenesis).
Hi,
yes you can :) Do not paraffin embed this defeats the advantage of frozen sections ie no antigen retrieval needed. I am attaching my protocol. As Michael said you will get much better morphology if your tissue is dehydrated through a sucrose gradient before embedding for cryo. Good luck
Nidul,
Your tissue must be cryo-sectioned (10 um?) adhered to subbed slides, or coverslips.Then you can apply a routine IF protocol. Hopefully your tissue was cryo--protected (sucrose) before freezing or you will have ice damage.
Good Luck.
Dear Nidul,
IF is just fine with the frozen tissue. But I would suggest to embed the thawed tissue in paraffin to get good thin sections in microtome, if you do not have the facility for cryosectioning as suggested by Delannoy.
I guess this should work. Please refer to the protocol post paraffin the following link.
http://www.rndsystems.com/ihc_detail_objectname_fluorescent_ihc_paraffin_tissue.aspx
Hi,
yes you can :) Do not paraffin embed this defeats the advantage of frozen sections ie no antigen retrieval needed. I am attaching my protocol. As Michael said you will get much better morphology if your tissue is dehydrated through a sucrose gradient before embedding for cryo. Good luck
I was just giving him an alternate suggestion iff the cryo technique is not available. Yes, paraffin is a very orthodox technique and it does require a few tedious steps that follow up after sectioning. In that case, cryo- is the best sought after technique.
BTW Caroline, thanks for the protocol share.
@Caroline Elizabeth Dunk
Thanks for the protocol, I will surely give it a try.
As others have already suggested do not thaw your tissue go ahead with cryotomy as it resolves the need for antigen retrieval. If I had fresh tissue I would still use crytomy for this very reason.
I might ask you to clarify though before going ahead on how long your tissue been frozen for and at what temperature?
Best Wishes
Adam.
Hi Nidul,
It is a real challenge finding the best method for your tissue. I have sectioned lots of different tissue types, and I would recommend trying a few different methods to see what generates the best image. More delicate tissue, eg. skin, can be quite difficult to work with when cryostated. I pop my tissue into OCT before freezing it for the cryostat method. I would really recommend that you try to section your tissue asap. I generally try not to leave my tissue in the freezer for more than 2 weeks for easy cutting. For long term storage, I would definitely recommend paraffin embedding it, then sectioning it with a microtome instead of a cryostat. The paraffin or OCT embedding of the tissue must be done before freezing, as someone previously mentioned above, otherwise the tissue tends to shatter when you cut it and you will get messy images, as the cells will be damaged from the ice crystals.
As far as generating a good IHC image goes, I have found that some markers are much brighter when I use frozen sections as opposed to tissue prepared with paraffin embedding and zinc fixation.
I hope you find a method which works well for you! Good luck! :D
in think on paraffin section and on frozen its not possible as tissue exhausted,,,,but alternatively Dukes mam protocol was good.
Hi, I have read the answers posted here but I was wondering about the tissue which has not been fixed before freezing. We have some frozen brain tissue (human) and is it possible to embed the frozen tissue as is into OCT and cut cryosections?
I have always used previously fixed tissue to cut sections and it has worked fine. But now I have some samples which were frozen without any fixation. I am not sure if the thin slice of OCT and tissue will stick well to the slide? Does the fixation of tissue have an effect on how well it attaches to the slide?
Also, is it okay to fix the section on the slide in formalin for 30 minutes? And then proceed with immunostaining?
Thanks
I think you will have to post-fix your sections, since under fixation will result is edge staining.
Try acetone -20 for 10 minutes.
In your case the fixation of the tissue sections is needed. We would do 4% paraformaldehide in PBS for no longer than 10 min at room temperature. You could use acetone as well but it depends very much on the conformational state of the protein epitopes after acetone treatment. Acetone interrupts the hydrogen bonds that stabilize the tertiary structure of the proteins. The antibody may not be able to recognize the protein after aceton treatment.
Hi Caroline Elizabeth Dunk,
If my tissue has been stored at -80C in 90% FBS:10%DMSO could I still perform IHC on these frozen (unfixed) samples using your protocol?
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