Staining for laser capture microdissection

[nnguyen]

Dear all,

I am trying to stain frozen inflamed liver sections (10 microns thick) for laser capture microdissection and subsequent RNA extraction. As you may know, the staining protocol must be quick, free of RNases and dehydrate the sections for efficient LCM. As a result, I am using the LCM staining kit from Ambion which utilises the cresyl violet stain for nuclei staining.

When the staining protocol works, individual hepatocytes and lymphocytes can be clearly seen. However, the majority of my staining results in this dark and brown cloudy like substance which looks like some sort of precipitate. The pics below illustrate my point and as you can see this precipitate clearly disrupts the interpretation and morphology of the section.

Can anybody out there help me in troubleshooting this problem?

The protocol is as follows after the section has been cut:
Fix in 95%, 75%, and 50% EtOH for 30-40 seconds each.
Apply the cresyl violet stain for 30 seconds.
Dehydrate the sections in 50%, 75%, 95%, 95%, 100%, and 100% for 30-40 seconds each.
Clear the sections by immersing the slide into one wash of xylene and then another xylene incubation for 5mins.
Air dry for 5mins and then LCM.

Kind regards,

Nam
http://www.immunoportal.com/modules.php?set_albumName=album01&id=Ambion_stained_section_of_RNA_Later_fixed_liver&op=modload&name=Gallery&file=index&include=view_photo.php

http://www.immunoportal.com/modules.php?set_albumName=album01&id=Ambion_stained_liver_section&op=modload&name=Gallery&file=index&include=view_photo.php

[richard03]

You may need better fixation before staining, for example, formalin or paraformaldehyde to fix the sections.

Another suggestion is to rinse sections in water briefly before cresyl violet staining.

[nnguyen]

Hi Richard03,

The fixation recommendations are probably not the best idea in terms of preserving RNA.

I have tried the water rinse but that wasn't able to remove the precipitate (if it is a precipitate).

[richard03]

Fixation is probably playing a key role here for the cresyl violet staining. So you may try a longer fixation with EtOH.

[nnguyen]

Please excuse my ignorance.

But what is the priniciple behind EtOH fixation? Exactly what is getting fixed? I don't quite understand what is getting fixed and how it works.

Thanks.

[CanuckPhD]

Etahnol (plus methanol and acetone) are simple fixatives of tissues and proteins. They are usually used to fix cryosections or cell smears. It is thought that these fixatives act to displace the water from the proteins, thus breaking hydrogen bonds, this in turn causes loss of tertiary structures. The proteins in the cytoplasm are coagulated but the nucleic acids are usually untouched.  It must be remembered that even the precipitated (i.e. denatured) protein can still be soluble. Perhaps the protein in your sections are not denatured enough and then are subjected to water. I assume as the nucleic acids are not precipitated by EtOH this is the reason it is used as a fixative in your protocol.

I noticed that you do not have a 100% EtOh step in your fixation. I would suggest that you add a 1 minute abs EtOH at the start. In addition make sure your slides are dry before you start, at least 30 minutes at room temp. (or will this allow RNAse to work?)

Good luck

[nnguyen]

That's a great explanation CanuckPhD. In terms of actually explaining the cause of the precipitate on my sections, your theory seems the most plausible one compared to other theories that I had generated!

As you had guessed correctly, I am cautious to let the sections dry at room temperature for 30 minutes in fear of activating endogenous RNases.

I will giving the 100% EtOH fixation an attempt. Thanks!

[Heidelberg]

Hey everybody,

Unfortunately, we´got now the same problems with our cresyl violett staining (and the same protocol) as described above?

Does anyone know, whether the 100% EtOH helps or are there any other suggestions?

Many thanks and best regards!

Christoph/Heidelberg

[nnguyen]

Hey everybody,

Unfortunately, we´got now the same problems with our cresyl violett staining (and the same protocol) as described above?

Does anyone know, whether the 100% EtOH helps or are there any other suggestions?

Many thanks and best regards!

I'm amazed somebody else has the same problem as me!!!

I resolved the problem by simply omitting the final xylene incubations. I believe there must be some sort of interaction by internal water in the tissue that could not be fully dehydrated and the xylene.

Omitting the xylene shouldn't give you any problems with RNA degradation and morphology. Keep the protocol as stated, 95% EtOH should suffice. Good luck with it and update me with some news, I'm keen to hear another user's opinion and results.

[Heidelberg]

Hey Nnguyen,

thanks for your quick reply.

We use the same staining protocol as you do.  We have modified it by addinng RNA-later for 5 secs and short washing in DEPC-PBS 1% before starting with rehydrating with etoh.  We have not used xylene, but nonetheless our protocoll worked perfect.

Curiously, when I started with the staining, we had the problems described above.
So I tried to exclude a lot:
RNA-later yes/no: no difference
PBS yes/no: no difference
air drying: no difference
xylene yes/no: no difference
keeping slides cool/warm: no difference
methanol before etoh: no difference
discarding all reagents and replacing new reagents: no difference.

Interestingly, suddenly our problems with bad staining stopped (whyever?!) and everything went well for 1 year.
Now, our staining quality has decreased suddenly again.  Even samples, which worked perfect in the beginnning, have acquired this "dirt" staining now.
I have no explanation, especially, since we excluded every new factor.

I keep on trying - maybe the problem will resolve by itself once again

Does anyone know, whehter this staining problem has a negative effect on rna-quality?

Best regards

[CellExtractionExpert]

If you have problems with contaminations and oxidized products in your stained samples you should use a very fresh solution of stain that has not come into contact with air or light. Regarding hematoxylin I know that it can be filtered to get rid of precipitate but the staining is less intense. If  There is a H&E staining Kit out there that addresses exactly this problem. Maybe you want to try it out. It's also cheaper than buying a new bottle of stain. (mmi H&E Staining Kit Plus) http://www.youtube.com/watch?v=sUcYwj9Hrks&list=UUdioRLq30FseYNy04819tig&index=1&feature=plcp