Hi, John. Use the fixative buffer as follows: Formalin         100 ml 

                                                                     Distilled water 900 ml 

                                                                     NaH2PO4       4 g

                                                                      Na2HPO4       6.5 g

I'm not sure -  it's for the first time I hear about this use of glyoxal. As I worked (and still do, from time to time) on insect histology, I can recommend you some hints concerning the fixation and the general technique involved.

But, I have to know if you want to do topographical or cytological studies, because the techniques are differentiated according to the dimmension of the studied piece (you can work from one nucleus to a group of cells, or from an organ to an entire insect).

As a general rule - the smallest of the insect detail you want to study, the most penetrant fixative you have to use. Also - the cytological fixatives are based on osmic and chromic acids, platinum and other heavy metals salts (uranium, palladium, etc.) and so on, while the topographical ones include formalin, ethanol, picric acid, acetic acid, sometimes mercuric chloride or potassium dichromate.

This, because the smallest the detail (e.g. nucleus), the fastest it degrades after death. So, if you want to study meiosis, you will want to work on a maxima of 2x2 mm / testis or ovaria piece. The conservation of the subcellular structures is not so important if you want to study the topography of the inner wall of the medium intestine (e.g.), you want to see the general disposition of the tissue strata, not the mytochondria in it - in this case a piece of 5x5 mm or even 1x1 cm works excellent.

You can use general technique manuals (Langeron, Gabe, Humason, Gray etc.), where you can find both the recipes and the uses for each fixative. If this can help you,I can send you by e-mail the books I have (all the abovementioned minus Gabe). Or, for any kind of help concerning histology or insect histology, let me know.

Best wishes,

Dani

We use 10% neutral buffered formalin, or seawater buffered formalin for routine histology, however, formaldehyde is now classed as a carcinogen and as a result there is reluctance by some field staff to use it (it seems they are happy to pour petrol, but not formaldehyde!). As one of my roles is laboratory team safety officer, I am looking at alternatives for field use, that is cheap to use, does not involve heavy metals (mercury) or picric acid (explosive) or chemicals that are difficult to transport or dispose of.  When used, it also needs to retain compatibility with traditional H&E slide interpretation. One of the few possibilities is glyoxal, so I wondered if others, faced with the same dilemma, have tried it. 

I beg you pardon. Only after posting my answer I checked your profile. If I will find something concerning the use of glyoxal as replacement for formaldehyde, I will be glad to help you.

Good evening, dear John Brian Jones,

the issue of using glyoxal as a fixative for invertebrates is not quite easy to respond at or with exhaustive depth, respectively. The use of glyoxal in human biopises (medical histology, as you stated in your question) MIGHT be successful, but isn't at all in reality... I am not aware of any Histology Lab in a Pathology Institute or Department (knowing that there might be some somewhere out there) using glyoxal for fixation of their (human) biopsies on a routine schedule. This is due to several disadvantages Glyoxal has in its basic chemistry (despite being a / the simpliest aldehyde, BTW: especially failing of Immunohistochemical stainings). For a first "orientation" I would like to point you to the following posts in another forum:

http://mailman.yale.edu/pipermail/nhcoll-l/2011-August/004195.html

(just in case you can not access, write in this thread and I'll provide the postings) where you can find (mostly) a collection of the disadvantages.

If you haven't found other results by googeling let me know via this thread. I have some files on in my collection of Fixatives (but unfortunately none of these dealing with invertebrates).

It would be helpful if you could let us know about the invertebrate species you want to fix with glyoxal... Insects for example, I think, will not be fixed well since

a) GL is no "strong" fixative (penetration depth and fixation process),

b) has usually a high specific density (over 1.0), so your specs will float on the surface of the solution for a long while, and

c) "an aqueous solutions must be buffered to about ph 4 to be

stable, and they must also contain a small proportion of ethanol, which catalyzes the reaction of glyoxal with proteins*)". Interestingly the*)/ another *) source. (cf.*) http://www.virusys.com/documents/Fixation%20and%20Tissue%20Processing.pdf , you'll find there some interesting facts on Glyoxal too) reports on "rapid fixation/cross-linking of proteins of specimens, faster than FA".

Hope this can be of help for now, best wishes and regards,

Wolfgang

Thanks for that reply, very helpful. We are a diagnostic lab, so we can get tissues from any aquatic invertebrate (usually molluscs), and bits of fish.

Hi, any tips on using glyoxal for fixating fish eggs (salmon) ? We are only interested in observing the cell nuclei after fertilization under magnification (not cross-sections or microscope). Using buffered formalin/acetic acid as well as Stockard's solution today, but looking for less "dangerous" alternatives. Can I as a first step try Stockard's by replacing formalin with glyoxal ? How about ethanol - is that necessary? I will do my own trials eventually, but just looking for pointers for a start solution

There is quite a literature on the use of solutions to clear fish eggs, for example, see Stoeckel, Joseph N., and Richard J. Neves. "Comparison of methods for viewing the germinal vesicle in fish oocytes." The Progressive Fish-Culturist 54.2 (1992): 115-118.

Stockards solution ((85% water, 5% glycerol, 5% acetic acid, 5% formaldehyde solution) (there is some variation on the proportions) probably uses the formalin as a preservative, and the glycerol as the clearing agent. If so, glyoxal may work as a substitute for formalin, but you would have to do some trials.  If it works - let us know!

Thank you John, although I feel like I hijacked the thread that was not the intention. I wasn't aware of the several versions of Stockard's out there. Thank you for the reference, that one was new to me - and probably very useful! I have ordered it from my local library. This should definitely get us going ! I will update you guys on the outcome - although perhaps it would have been more useful as a separate topic ?

Addition in brief (concerning/regarding  particularly Glyoxal [ethane-1,2-dione] as fixative...

not for   "only to clear fish eggs":

@ John: have you found meanwhile specific literature on glyoxal-fixation? E.g. 

 by RAMOS-VARA & MILLER, 2013, can be found at: http://vet.sagepub.com/content/51/1/42.full.pdf+html

(just to cite from that paper: 

>

Another publication regarding fixatives/fixation matters (comparison,evaluation) would be also (OPEN ACCESS):

Thank you for the references, they are useful additions to the debate.  Its becoming clear that when we have the time :-( we will have to do our own comparison experiments.