[genzeryk]

Hi Guys,

This is my first post here and I hope you could help me. So far I managed to solve my questions on my own but this time I decided to ask for some help as the results of wrong actions can be catastrophic for my research project.

In biological sciences 4% formaldehyde is routinely used to "fix" biological samples. The reaction goes like that:

  [protein]-H + HCHO --> [protein]-HCHOH
  [protein]-HCHOH + H-[protein] --> [protein]-(CH2)-[protein] + H2O

This means that proteins form covalent bonds and everything gets nicely bridged preserving the structure of cells, tissues, etc.

The protocol that I have to use includes incubation of the tissue in an aqueous 4% solution of ammonium thiocyanate (NH3+ SCN-). Because my tissue is very fragile, it is absolutely critical to minimise the number of processing steps. I was wondering if the remains of the thiocyanate can somehow interfere with the fixation reaction with formaldehyde (i.e. if I do not wash it thoroughly away before adding FA).

My question is then: Can ammonium thiocyanate react with formaldehyde in aqueous solution or anyhow interfere with the cross-linking of proteins by formaldehyde?

Thanks so much,

Genzeryk

[Honclbrif]

Just out of curiosity, what's the thiocyanate doing? Removing metal ions?

Formaldehyde crosslinks proteins via amino groups, so adding extra ammonia could very well cause a problem. You may be able to get away with using excess formaldehyde. While some will react with the ammonia, you'll have more left over for crosslinking.

If you've got plenty of material to work with, could it hurt to try it with and without the wash? Otherwise, if your samples are valuable or sensitive in some say, I'd recommend asking someone who knows more about tissue fixation, or hitting the books on this one.

[Mitch]

Expanding on Honclbrif's point. It would be prudent to add a PBS washing step between the ammonia thiocyanate step and the 4% formaldehyde to avoid proteins binding to free ammonia.