[ChromogenHaze]

I apologize in advance if this is the wrong place for this. Also, I know little about chemistry but I am trying to really understand tissue fixation (I work as a Histotech in a lab). There is a good chance I'm not even asking the right questions, but if someone could point me in the right direction that would be awesome!

From what I have gathered, formaldehyde has been used for over 100 years as a fixative, but what is actually happening on a chemical level is not well understood (at least by those mostly interested in fixation). I know formaldehyde is what forms crosslinks with proteins in the tissue, thereby "fixing" it, but this takes a quite a long time because there is so little formaldehyde actually available.

The formaldehyde we use for fixation is supplied in a 40% aqueous solution (often called formalin), which results from bubbling formaldehyde gas through water until no more will dissolve. However, the formaldehyde is rapidly hydrated to form methylene glycol, and the equilibrium is waaaay in favor of methylene glycol: 99.06:04 (i.e., 2499:1)¹. As that tiny bit of formaldehyde forms bonds with the tissue, more formaldehyde forms from the dissociation of the methylene glycol. Unfortunately, this means at any given time there is very little formaldehyde for binding, and why tissue fixation with formaldehyde takes so long.

My question, finally, is why does this happen? And, would there be a way to tip that equilibrium in favor of formaldehyde, thereby making more immediately available to bind with tissue?

I probably don't have enough of a background to understand exactly whats happening, but any help would be appreciated.

¹https://www.sciencedirect.com/science/article/pii/S0273230014000531

[Borek]

My question, finally, is why does this happen?

I am afraid the only answer is "because thermodynamics favors this reaction".

From what I remember formaldehyde undergoes several reactions, including some polymerization. Most likely shifting the equilibria is possible by changing pH of the solution, but I doubt it wasn't tested in the past, it is used for fixation for well over 100 years.