Keeping the tissues in 4% PFA (or 10% formalin) can result in over-fixation or the extensive cross-linking of the antigen proteins leading to a weak or no signal when performing immunostaining (something that you already observed in your testing). In general, 8-24 hours of fixation is plenty for most of the tissues and the best way to preserve the tissues afterwards is to keep them in 70% ethanol. I and a colleague of mine used to work on xenograft tumors and our study designs also required us to collect tissues at different different time points. Saving fixed tissues in 70% ethanol is something that we employed in such situations without any issues,     

Dear Katharina Nöske :

Yes it is. as Subhash Chander Gangar earlier. Also some people use PBS for 2-3 days and for more than 2-3 days they keep in ethanol 70%.

Dear Subhash Chander Gangar :

May i know did you wash first with PBS and then kept in Ethanol 70% or straight you transferred to Ethanol 70%?

Besides, may I know where you kept ethanol 70% ? RT or 4 degree?

according to our pathology and histology department experience as a 30 years expert, one year 4%PFA/PBS is also fine, the point is that you should just manipulate the protocol a little bit, git rid of Sudan black don't use it at all, for DAPI the only vectshield is enough to do washing of all steps in PBST or TBST and dilute antibody in TBST/BSA 1% . over fixation can't made problem but under fixation would make problem. Good Luck

I do 24hrs post-fixation in 4% PFA and keep the tissues in PBS+ 0.05% sodium azide. Sodium azide being a preservative helps you to keep your tissue very longer like even 6 months and more. Our tissues are brain but I guess this procedure should work good for even other tissues. My immunofluorescence is also good.  

Hello Mostafa,

As already mentioned above, fixation of tissues depends mainly on two factors: the type of tissue and the thickness of tissue. Try and find out which fixative works best for your tissue. However, 24 hours fixation with 10% neutral buffered formalin or 4% paraformaldehyde seems to work well for most tissues.

After the course of fixation, it is essential to wash the tissue with 1X PBS as it helps to get rid of the fixative. Both over or under fixation can lead to problems with your downstream steps. After washing, you could store the tissue in 1X PBS or in 70% ethanol. Post fixation, tissues can be maintained in 4 degrees or in room temperature. I, however, prefer to keep my samples in 4 degrees.

Also, fixation depends on the type of sectioning that you are planning to do with your samples, that is, if you are not going for whole mount staining altogether. If you are going for paraffin sections, the aforesaid method is alright. However, if you are going for cryo sections, you can directly go for embedding and flash freezing your samples with OCT, in liquid nitrogen. After obtaining the cryo sections, you could directly fix the sections on the slides for around 30 minutes in 4% paraformaldehyde or 10% NBF. Prior to fixation, you would need to wash with 1X PBS to remove the OCT. After fixation also, you would need to wash with 1X PBS to remove the excess fixative. Such sections need to be stored in 1X PBS until processed further.

Hope that helps and good luck.

Dear Masoume Majidi zolbin and Samyutha Rajendran :

Thank you for your useful information.

Dear Atreyi Biswas:

Thank you for your deep and useful guideline. I think I will try both methods (PFA and Cryo) at different fixative time and see which one will be ok.

4% PFA for 24 hrs is only over fixation, and it is not reccomended for routinary immunohistochemical analysis.This because both the time of fixation and storage in ET-OH impair tissue antigenicity.

Please take a look at various immunohistochemical papers appeared in all the authoritative journals.

I would fix small pieces of tissue for 2-4h in 4%PFA, then wash them thouroughly in PBS - at least a couple of changes, 30min each, then keep in 70% ethanol.  

Yes ! Elena your fixation protocol is OK.In fact I performed 4% PFA fixation for few hours of the gill, swimbladder, lung , gut and skin tissues of fishes by several years.I got very nice confocal images using double labeling procedures with several antibodies to neuronal markers and nNOS.

Best

Giacomo

hello, I am planning to collect several organs from mouse ( kidney, lung, tumour tissue, spleen). Could I fix them in PFA 4% , overnight? Post fixation, they will go through couples of washing of 1XPBS and stored in 1X PBS. How long can they be stored in the fridge?

Thank you.

I agree with Elena's protocol. Brain is such a dense tissue that overfixation will not hamper the staining; however for small and delicate tissues, 24h in PFA is more than enough. It's best to make either paraffin or OCT blocks and store it for future use

After PFA fixing,the tissue slices are to washed in PBS two times depending on the fixation hours,and allow these to be dehydrated in a graded et-OH ethanol series before paraffin embedding