Following.

[LONG]

Just to complete the data for the recipe as given in the first really "meaningful" (= but 2nd) reply by Simon A. Dingsdag

According to the website / Catalogue of EMS's fixatives -to be found at: https://www.emsdiasum.com/microscopy/products/chemicals/fixative.aspx

[quote:]

"EMS is proud to introduce an alternative to Formalin and Formaldehyde in many different applications.

Our unique kit comes premixed and measured and ready to use. The kit consists of 2 vials: Solution A & Solution B (Absolute Ethanol, Glyoxal Solution, Acetic Acid and water).

Instructions:

Add solution A to solution B. Vortex the two solutions together and bring to a pH of 4.5 with the addition of NaOH 1M. The solution needs to be used immediately once mixed.

References

• Sabatini DD. et al (1963) "The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation". J. Cell Biology. 17:19-58
• Janigan DT. et al (1965) "The effects of aldehyde fixation on acid phosphatase activity in tissue blocks". J. Histochemistry. Cytochemistry. 13: 476-483
• Dapson RW. et al (2006) "Glyoxal fixation and its relationship to immunocytochemistry." J. Histotechnology. 29: 65-76
• Dapson RW. Et al (2007) "Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval" lnforma healthcare, Dapson & Dapson, LLC
• Richter K. et al (2018) "Glyoxal as an alternative to formaldehyde in immunostaining and super-resolution microscopy" The EMBO Journal. 37: 139-159.

[End of quote]

Since we do not know exactly about the volumes of vial 1 and vial 2 (and the explicite proper proportion needed) one cannot calculate 'smaller volumes' for the two components provided by EMS (it would be able if both vials contained solutions each with the same volume and they are to be mixed together 1:1 ...but see below BUSSOLATI et al. 2017!).

The recipe as given by Simon A. Dingsdag

Paraformaldehyde (PFA) and glyoxal preparation :

RICHTER et al, 2017(2018)

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy.

DOI 10.15252/embj.201695709 | Published online 16.11.2017 The EMBO Journal (2017) e201695709 cf.: http://emboj.embopress.org/content/early/2017/11/16/embj.201695709

For sure:

An interesting stuff with regard to fixing properties (not only: "morphology in general, Histology, Histochemistry, IHC, EM" etc. but also regarding the safety issue of "carcinogen PFA/FA-solution and vapours" ), so

perhaps worth reading too:

(Q by John Brian JONES 5 years ago in RG):

https://www.researchgate.net/post/Has_anyone_used_glyoxal_as_a_substitute_for_formalin_when_fixing_invertebrate_tissues

http://www.virusys.com/documents/Fixation%20and%20Tissue%20Processing.pdf

BUSSOLATI et al, 2017:

'Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples'

Article Acid-free glyoxal as a substitute of formalin for structural...

(NB: delete 'underscore characters' __ in between 'https' and ' ://...' before linking to website for the original article @ :

https__://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182965

[ Quote: ]

Materials and methods

Reagents

Glyoxal (40% in water) and basic ion-exchange resins (Amberlite® IRA-400 chloride form and Amberlite® IRA-67) were all purchased from Sigma (Milan, Italy). Addition of commercial glyoxal (40% in water) to a 0.1 M phosphate buffer pH 7.3 at a final concentration of 2% resulted in a drop of pH to the acidic side and this solution was in the range of pH 4 after 6 months. To deprive glyoxal of acids and thus to obtain Glyoxal Acid-Free (GAF), 250 g of the strongly basic ion-exchange resin were moistened with de-ionized H2O, then activated by a short wash with NaOH 1 M. Following washes in water to remove sodium hydroxide, 150 ml of glyoxal were added. The resin was allowed to act for 30 min at RT and then removed by passages through a filter. The resulting, water-clear liquid, whose pH is around neutrality, represented 40% glyoxal deprived of acids. We adopted a 2% solution of GAF as the fixative of choice. The 2% solution was selected by referring to the same amount of aldehyde as present in 4% formalin.

The resin, once used to remove acids from glyoxal, can be re-generated (with a bath of 1M NaOH) and re-used.

A 2% GAF solution in 0.1 M phosphate buffer pH 7.3 is stable for a few weeks then gradually undergoes oxidation producing an acidic reagent with sub-optimal fixation properties. To overcome the stability problem, we produced a stock solution containing 20% GAF in 50% ethanol (Carlo Erba, Milan, Italy) added with 0.1 g insoluble calcium carbonate (Sigma-Aldrich) in 100 ml of the solution (stock solution). The final (working) solution employed as GAF fixative was obtained by diluting the stock solution (exempted of calcium carbonate) 1:10 in 0.11 M phosphate buffer pH 7.3. [ End of quote ]

Article: "When Tissue Antigens and Antibodies Get Along: Revisiting the Technical Aspects of Immunohistochemistry—The Red, Brown, and Blue Technique"

J. A. Ramos-Vara and M. A. Miller

Veterinary Pathology, 2014, Vol 51(1) 42-87

(C) The Author(s) 2013

Reprints and permission: sagepub.com/journalsPermissions.nav

DOI: 10.1177/0300985813505879

vet.sagepub.com

p. 47: quote

"Glyoxal, the Ideal Formalin Substitute? Glyoxal is a dialdehyde that resembles 2 back-to-back formaldehyde molecules.76

Whereas formaldehyde forms a single hydrated species (methylene glycol), glyoxal has several hydrated forms, the most common of which is 1,3-dioxolane.76 One advantage of glyoxal over formaldehyde is that it does not emit vapors. In addition, glyoxal has poor reactivity toward most end groups and forms addition compounds only with arginine, lysine, cysteine, and the single a-amine group of proteins, whereas formaldehyde reacts at room temperature (RT) with almost all end groups that contain nitrogen or oxygen to form singlecarbon

adducts.76

In comparison to formaldehyde fixation, glyoxal fixation is faster with minimal or no cross-linking, rendering antigen retrieval unnecessary for many antigens.

Although a special antigen retrieval solution with high pH can be used, standard antigen retrieval procedures in glyoxalfixed tissues may be ineffective or even deleterious.76

The perceived advantages of glyoxal were disputed, however, in a quantitative image analysis study in which formaldehyde was considered superior in terms of morphologic preservation and IHC signal.59" [end of quote]

R.J. Buesa

Methods in Pathology / Histology without formalin?

Annals of Diagnostic Pathology 12 (2008) 387–396

p. 392 (quote):

"....Summing up, all Glyoxal-containing substitutes, although

they eliminate the dangers posed by formalin, are no match to its

morphological and preserving qualities and, except for Safe Fix

II, have not been evaluated regarding the nucleic acids preservation,

the second problem to be solved by the substitutes." [end of quote]

DAPSON 2007: https://www.tandfonline.com/doi/abs/10.1080/10520290701488113?src=recsys&journalCode=ibih20

Article Glyoxal fixation: How it works and why it only occasionally ...

PS-Disclaimer: No affiliation to or financial interest in any of the mentioned companies....

Apologies for lengthiness and spelling errors if existent....

Dear Denis Korneev , thank you for your interest and kind recommendation!

To complete the information finally:

Giovanni BUSSOLATI

(who is First author of:

BUSSOLATI et al, 2017: 'Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples')

IS THE HOLDER OF A ( not exhaustive! ) PATENT :

cf:https://patents.google.com/patent/WO2017140596A1/en

More of his innovative activities one can find:

eg: https://patents.google.com/?inventor=Giovanni+Bussolati&oq=Giovanni+Bussolati

or, only as one out of many more:

https://patents.google.com/patent/US20190049348A1/en?inventor=Giovanni+Bussolati&oq=Giovanni+Bussolati

Best wishes and regards, W.H.M.

Dear Wolfgang H. Muss , thank you for sharing the information. I have never used Glyoxal, but all "alternative chemistry" for EM (new fixatives, stains, etc) is an interesting topic for me.

Cheers,

Denis

Dear Denis Korneev, I have tried glyoxal only twice in experiments with human carcinoma cell line MDA-MBB-231 and with protist Entamoeba histolytica and the results were bad. I performed TEM imaging and the whole cells looked extracted.

Hopefully you will get better results. I will try the experiment one more time at least to get better results but now I am quite sceptical... .

Best,

Lenka