inking question

[sarephina]

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Just curious... The solution that we use to "fix" the ink on specimens is ethanol, and sometimes acetone. Does anyone know why we use these solutions? Are there other solutions? Different types of ink? Is there a chemical reaction with the ink, or does it just help the ink dry faster? I've noticed the ink kind of clumps up when I mix it with ethanol... nobody I've asked so far seems to know... I can find info about staining dyes in practical histology books but haven't seen anything about these pesky inks yet...

 

[sirenomelia]

There are four-five major groups of fixatives, classifiedaccording to mechanism of action:

Aldehydes- include formalin and glutaraldehyde.
Formalin-fixed tissue is fixed by cross-linkages formed in the proteins that preserves protein structure/ antigenicity so formalin fixed tissue is suited for immunoperoxidase studies. Formalin penetrates tissue well, but penetrates slowly- 1mm/hr. Glutaraldehyde penetrates quickly, but very poorly overall and does alter protein structure so is not ideal for IP staining. It gives best overall cytoplasmic and nuclear detail so is suited for EM.

Mercurials- (B5, zenkers) fix tissue by an unknown mechanism. These fixatives penetrate relatively poorly but are very fast and preserve excellent nuclear detail. Their best application is for hematopoetic and reticuloendothelial tisues.

Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause tissue brittleness. However, they are very good for cytologic smears because they act quickly and are cheap.

Picrates include fixatives with picric acid. Foremost among

these is Bouins. It has an unknown mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as much hardness.

http://khairul.forsciedu.com/Personal/Pdf/Advanced%20histotechniques.pdf​

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[Sulfinator]

Formalin-fixed tissue is fixed by cross-linkages formed in the proteins that preserves protein structure/ antigenicity . . .

It should probably be pointed out, though, that the preservation of antigen recognition is variable and depends on the particular antigen/antibody combination you are talking about. Obviously, the antibodies routinely used in pathology labs have been selected as those that tend to work well with formalin-fixed tissue; but, as a more general principle, some antibodies do work and others do not work in this context.

 

[cjw0918]

We used to use Bouin's to help make the ink stick, but it's (supposedly) highly carcinogenic so we switched to vinegar. Works as well.

 

[Ombret]

I suspect these ink fixatives are not doing anything too specific, because there are different types of dyes and they all seem to respond similarly to the fixatives. My guess is that anything that denatures or fixes the tissue is going to promote forming some noncovalent interactions between the dye and tissue.

 

[KCShaw]

I think this is a reasonable question. As far as 'any old fixative' doing the trick to cause ink to stick and stop being runny goes, personally I had trouble if formalin got on my inked specimen before it was ready, but maybe that had more to do with the water which was diluting it. I used Bouins as a mordant to 'set' the ink, but I confess I don't recall how it or other mordants work in this setting; I seem to recall it only really worked with certain ink colors and not with others. I would also spend time drying and trying to get as much tissue grease off of the specimen as possible prior to applying ink, which sped the whole process up, but isn't easy to do with every specimen.

I note that some searches reveal Bouins to technically not be a tissue mordant as it's not metal based, but those discussions seem to be about tissue staining protocols for slides, not gross inking. Whatever the proper terminology, it appears Bouins &/or vinegar are relatively common for this purpose.