Dear David,

In our hands we get a better staining of membranes using ferrocyanid compared to ferricyanid. Solution of OsO4-ferrocyanid my have some brownish precitpitate, which does not really disturb, whereas OsO4-ferricyanide solutions are clear with an amber colour.

We use the following protocoll:

Fixation by immersion in Karnovsky´s fluid (2,5% glutaraldehyde, 2% paraformaldehyde at 4 C for 24 hours

Afterwards the small specimen are brought in 1% aqueous OsO4 and 1,5 % potassium ferrocyanide (Sigma Aldrich) for 1-2 hours.

Regards, Daniela

David, since it is years ago I have worked with osmium +/- ferricyanide (OsFeCN) or osmium +/- ferrocyanide I would like to honestly ask if you have had at least a short look into the older literature related to these modifications of primary/ secondary fixation, for example:

HEPLER PK,1981*: The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining*, in Eur J Cell Biol. 1981 Dec;26(1):102-11. cf.: https://www.ncbi.nlm.nih.gov/pubmed/6173219

or

HORN JW et al, 1996*: Transmission Electron Microscopic Comparison of Osmium vs Osmium with Potassium Ferricyanide Secondary Fixatives and the Impact of Secondary Fixative Temperature on Tissue Preservation/Contrast Quality*. Proc Microsc Microanal 1996,p.910-911 (cf.e.g.: http://jglobal.jst.go.jp/en/public/20090422/200902129870696595)

or:

HJALMARSSON C et al, 2004*: "Electron microscopic evaluation of the endothelial surface layer of glomerular capillaries" , Microvascular Research 67 (2004) 9 – 17

cf.: Article Electron microscopic evaluation of the endothelial surface l...

or

LANDERS St C, 2011*: Microscopy Protocols: "Improving TEM Fixation with Additives" (i. e. 1,5%GA+0,2%TA & 1%OsO4+1% K-Ferrocyanide, oocytes) Micr.Today 19 No1, 40-42 Jan 2011, cf (free access): https://www.cambridge.org/core/services/aop-cambridge-core/content/view/EC5C8BFFACD66004AC541E2B9F132AE0/S1551929510001173a.pdf/improving_tem_fixation_with_additives.pdf ,

and so on...

As usual, it [special effects of the two combined fixatives] will depend on the task of the study as wll as on combination of not only Os-potassium ferrocyanide but sometimes further addition of components (like tannic acid) might produce entirely different results in terms of contrast or e-density...(and other properties as well). Therefore your question might be a bit deceptive ('misleading' ) because the effects of both the above mentioned components in different 'organ systems' might be different and at least have to be tested once by a comparison of consecutive trimmed blocks of tissue(s) which are processed more / less equally -except the addition of either K-ferri- or K-ferrocyanide to the Osmium- solution.

NB: * means I have the pdf's in my private e-library collection (ask [email protected] or send an RG-message if you are in need of).

MY best wishes....and good luck!

In case it can be helpful, a Karnovsky fixation with paraformaldehyde, glutaraldehyde, cacodylate buffer and Ca Cl2, before fixation with osmium, has given us excellent results in microalgae (photobionts) of lichen thallus

Dear Carmen, thank you for your comment.... but - as I understand it - David is aware of the necessity and "the benefit" of proper primary fixation for TEM studies. And, that quality of TEM-staining [similar to an impregnation after primary fixation) might depend on a "first class fixation" prior to the use of potassium ferricyanide or ferrocyanide with the swecondary fixative, osmium tetraoxide. For the task David is thinking about it would be - perhaps - also interesting to think about methods using (low molecular weight) tannic acid ....

Just for convenience I add here an abstract including a personal compilation of what W. F. NEISS presented in an oral lecture during the 4th Annual Work Meeting of the German "Anatomische Gesellschaft (Anatomical Society)" 1983 (!) in Wuerzburg, Federal Republic of Germany (now Germany) where that author reported about his experiments on OsO4-K-ferrocyanide secondary fixation and the impact of i) primary fixative and ii) the effect of molarity/percentage of the ferrocyanide on staining of cellular organelles and compartment textures... Regards, W.M.

I very much agree with your comment and I appreciate your valuable input