I've used 1% OsO4 in 0.1-0.2 M cacodylate buffer for the past 30 years on everything from plants, animals, bacteria etc.. I've never heard of this issue being a problem. I'm usually pretty happy with the results.

Colin, would it be possible to copy and paste the respective text (section osmication/osmium  from Barbara Gabriel's book 'B.E.M.' just to be able to find own opinion?  I always used (for at least 30 years of diagnostic work PO4-buffered aldehyde and osmium fixative solution....and there are some reports on the superiority of "Millonig's" Na-Na-buffer at pH7.0-pH7.2 (I could provide recipes, figs. and lit.sources /references, if you are interested). The use of cacodylate buffer in routine diagnostic specimen preparation was stopped after a relatively huge increase of prices for 100g/500g in Austria in the 1980ies. The remaining / the amount  which was left over at that time point  was used up peu a peu only for very special specimen preps. Best wishes and regards, WM.

I stopped using buffers a while ago and did not notice any difference. OsO4 works fine with plain distilled water. When I did used buffer it was cacodylate.

Hi Colin,

I didn't read that book, but I can tell you I've always used cacodylate buffer 0.075M for OsO4, and I'm pretty ok with results (I mostly work with plant tissue)! 

To all: Okay, whew, I was worried. Her book is very complete otherwise, with plenty of info on embedding media, fixatives, and so forth. It's just that was a very odd passage and perhaps there was something to that I had missed.

Wolfgang: The book was written in 1982. So there might be something to that! Since I can't get a decent link, I'll just enclose a snapshot of the text. Thanks for the offer of the other buffers! I'll take you up on that reference for Millonig's! Monobasics shouldn't have the precipitation issues of dibasics, right? Can't promise I'd use it, though, since so far cacodylate has given me the best results out of all the buffers I've tried.

To ResearchGate: Why are my topics now about Dickensian London and Phosphates? That's not even close to what I had set Friday. Wh-what does any of that have to do with what I typed? What do they even have to do with each other? =P

Dear Colin, thank you so much for your kind reply and also the snap-shot out of the text.  While I would like to answer quickly about what was coming to my mind when seeing the text (both two or three sentences with reference quotations) - i. e. B. Gabriel perhaps reasoned about an incompatibility of Os-ions / metal and the As-ions / arsenic components in the sodium-cacodylate (this might be why she directed the reader to the use of the "inert" sym-s-collidine buffer – which in 1982 was quite modern to use - and also mentioned the incompatibility with uranyl-actetate) - I guess -this today might be one myth more about ‚cooking‘= empirical recipes in EM (though I was not able to prove that by myself, nor have I found or - at least - in my mind a reference quoting / considering / speaking explicitely (about) such an incompatibility of the mentioned ions in solution. But now that I have seen that statement in B. Gabriel's book I shall be alerted when reading  "old(er) recipes" when they come to my attention in the future. If I find something I shall post it here.

Regarding the Millonig's Na-Na-buffer, I shall send as soon as possible my files to you (please write an e-mail to [email protected]). For the interested reader here I would like to reveal the "nature/principle" of the buffer, since the respective buffer solutions (they are mentioned on RG already) are not ‚monobasic‘ phosphates, but instead, mono- as well as dibasic sodium-phosphate are mixed together at a certain molarity (resulting finally in 0.1M or 0.13M ) and a pH which can be chosen in the (roughly spoken) physiological and effective range between 6.5pH and 8.0pH. The "precipitation" issues you claimed above for the < dibasic phosphate > [I guess or think about the so called "salt & pepper-artifact]  is only a result of too harsh (= too rapid) dehydration by „higer“ concentrated alcohols [you practically could/can test that by mixing phosphate buffer with –say – 75% EtOH….you’ll have a precipitation like adding CaCl2 to phosphate buffer solution]):  If working with PO4-buffer during and after osmication (i. e. washing with 0.1M PO4-buffer after the application of the OsO4 solution) you should NOT start with 70 or 75% EtOH but only with 50% EtOH instead. Also, you shouldn't dehydrate in only 3 steps  [as this most of the time is done in HISTOLOGY:  70/75%, 90-95%, 100% EtOH] but 50, 70,80,90,96,100,100% would be fine. This all said only in case you PREFER using PO4 as your fixative vehicle and therefore also washing in PO4-buffer(s) in between GA-fix and OsO4-postfix as well as after the latter. As was mentioned by Vladimir, one certainly can postfix in only aqueous OsO4 (= in A. bidest), (the reason of which is out of focus here/insignificant, one optional washing step in A. bidest afterwards) and then start the dehydration in 50% EtOH or 70 % EtOH. My favorite processing - regarding the outcome of 'morphological' preservation of structures - always was to adhere to the more time consuming, multiple and hence smaller (%) steps of EtOH concentrations -  because elution on the one hand and "alcoholic precipitation" of previously not 100% fixed tissue components was not that harsh as with only 3 dehydration (%)steps. Looking forward to read from you in my private mail.... best wishes and regards, WM.

Hi!

Thank you all very much for this discussion, as it solved 2 riddles that were in my head for a long time.

First: I did routinely use buffered osmium teroxide, and for some years the default buffer was Na-cacodylate, and I never had problems with osmification because of the buffer. Some time ago, I switched to HEPES buffer, because the effort to safely discard of the As outweighs the price difference in my eyes, and this also worked fine. I only heard rumors that uranyl acetate does not work with every buffer, but this I use with unbuffered ethanolic solutions.

Secondly, I recently tried unbuffered aequous osmium tetroxide solution, and I, too, did not notice any difference, which does not surprise me, given the fact, that the membranes should already have lost every osmotic activity.

I, too, routinely go to the effort of multi-step dehydration, even going as far as using 3x5-10min per step instead of 1x20-30, and including a short, 5-10min step for both 30% and 50% before starting with 70%. 

Thank you all very much for your contributions. Discussions like this help a great deal in sifting through the numerous myths connected with TEM, and to reduce the complexity of protocols without sacrificing structural preservation. Usually, one person alone simply has not the time to test all the involved parameters systematically, so let´s share experiences whenever possible.

P.S.: Glass knives. Oh, yes. Well, if it works, it works. Problem is only if you have to change knives and can not afford to loose material...

[Apologies for lengthiness]

Dear Paavo, thank you, also for your great and acknowledging statement.

I would be interested in the (your) recipe for the HEPES -buffer YOU USED (stock-solution / working solution, kind of tissue) for some reason....(especially molarities, and if you had measured already, their mosmol & pH-values).

Regarding the possible use of unbuffered aqueous OsO4-solution because of completely lost osmotic activity of membranes was a concept already in the 1980ies if I remember correctly.  

Regarding precipitation of PO4-ions due to harsh dehydration (in only 3 steps) starting e. g. with 70-75% EtOH: not only such treatment would result in coarse, but also finest precipitation of phospate ions (which  - in turn - impresses with the so called "salt & pepper"-effect  [appearing not only after staining ultrathin sections routinely with UO2Ac & lead citrate but also after en bloc UO2-acetate fixation / staining {see **) below}: citation: „Specimen that have been stained en bloc using UO2 Ac can precipitate phosphate onto the block face [Ref.# 67], therefore it may be necessary to substitute TRIS-buffer for DPBS.“ ]).

When using phosphate buffer as washing solution (e.g. after PO4-buffered osmication)  prior to dehydration you MUST use Ethanol lower than 65% concentration to prevent immediate precipitation of the phosphate ions in the specimen.

When UO2-Acetate was used as a primary / secondary or - last but not least - as en bloc secondary or tertiary fixative and contrasting agent it  is / was necessary to (eventually) use the (inert) malonate (maleic acid) buffer sequence [*) see bottom note]   for washing the specimens prior to incubation in malonate-buffered UO2Ac-solution (NB: @different pH ). That should prevent the precipitation of phosphates and uranyl-acetate too....(recipes for that, e. g. prior to enbloc postfixation or even for improving the "uranaffine reaction for - non specific- localization of NSG's=neurosecretory granules in tissues" since in fact the method is localizing ATP in the granules -   I have saved in my files).

You are right with / in also demanding "simplification" of protocols ("…reducing complexity of…."). This might not be that easy though as long as - IMHO - the interdependence of chemical behaviour of used substances and solutions vs. tissue components are complex and perhaps sometimes not known or established well.

But if you - just only as an example - have a look into the so called "Nature Method Protocols(C),(R)" [if you are a lucky subscriber]  or eventually also into CSH-Cold Spring Harbor(C),(R) Protocols   [e. g., cf.:  cshprotocols.cshlp.org/site/recipes/nav_a.dtl]  you might learn about how to stipulate and govern "precision of steps needed" [RE: "certification and SOP's"],  and / but also sometimes a hefty dose of ignorance by authors narrating "wishy-washy"  "inherited < knowledge > "  .  

Regarding your problem with "glass knives" ....I used glass knives for a long time (and in the 1970ies &1980ies made them by myself using an LKB knifemaker 7800B for serial sectioning of 2 µm thick brain semitghin sections)....  but --  calculating the time needed for making them, taking into account the overall costs for the glass strips, the support material (cleaning thoroughly, tapes, dental wax, safe-keeping boxes, etc., and - last but not least - all those knives which had to be chucked away because they weren't good enough  --  after two or three years of drudgery in my profession afterwards  lead me to grumble about the situation and the outcome,  and finally it prompted me to contact then my (former) diamond knife manufacturer (yes, you are right: it might be pricey  first, but in the end it might be easier and more straightforward to achieve excellent section quality (reproducibility) and most of all, no problems losing precious material due to necessary change of i) glass knife edges' position or i) changing your glassknife at all...

Hope you find this helpful anyway....Best wishes and regards, WM..

Footnotes:

*) Concerning Maleate - Maleic acid buffer in CSH-directory:

Maleic acid buffer:  Cold Spring Harb Protoc; 2008; doi:10.1101/pdb.rec11524 [Full Text: http://cshprotocols.cshlp.org/cgi/content/full/2008/11/pdb.rec11524?text_only=true]

Maleic acid buffer (5X): Cold Spring Harb Protoc; 2009; doi:10.1101/pdb.rec11875 [Full Text: http://cshprotocols.cshlp.org/cgi/content/full/2009/8/pdb.rec11875?text_only=true]

Maleic acid buffer for sponge tissue (MAB): Cold Spring Harb Protoc; 2008; doi:10.1101/pdb.rec11541 [Full Text: http://cshprotocols.cshlp.org/cgi/content/full/2008/12/pdb.rec11541?text_only=true ]  

Uranyl acetate (0.05%) in 100% methanol: Cold Spring Harb Protoc; 2008; doi:10.1101/pdb.rec11366 [Full Text: http://cshprotocols.cshlp.org/content/2008/6/pdb.rec11366.full?text_only=true]

Uranyl acetate (saturated) in 50% methanol: Cold Spring Harb Protoc; 2008; doi:10.1101/pdb.rec11365 [Full Text:  http://cshprotocols.cshlp.org/content/2008/6/pdb.rec11365.full?text_only=true  ]

 ==> What do these protocols say? 

 vs.  (only as examples):

**) HYATT and EATON in their 1992 book: Immuno-gold Electron Microscopy in Virus Diagnosis and Research:  find: Williams and Faulkner in Chapter 7.: They define: „Uranyl acetate en bloc staining (= e.g. at p.195f), „Embedding pepper“ by uranylacetate en bloc staining of specimens;   cf. p. 208:

Precipitation of PO4 / Uranylacetate in Immunostaining; cf. Section  IV. Immunostain Techniques, p. 209:  citation: „Specimen that have been stained en bloc using UO2 Ac can precipitate phosphate onto the block face [67], therefore it may be necessary to substitute TRIS-buffer for DPBS.“ End of citation

Ref.# [67] = Berryman MA and Rodewald RD: An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes. Histochem.Cytochem.38, 15, 1990 [cf.: https://www.ncbi.nlm.nih.gov/pubmed/1688894  or Original article pdf: http://journals.sagepub.com/doi/pdf/10.1177/38.2.1688894 :                                   citation:  „To remove phosphate ions, tissue was rinsed with cold 0.1 M maleate buffer containing 3.5% sucrose, pH 6.5 (4 x 15 min), and then was post-fixed for 2 hr at 0°C with 2% uranyl acetate in sucrose-maleate buffer (final pH 6.0). Additional vehicles that were used with 2 % uranyl acetate included distilled water (final  pH 4.0), 0.05 M maleate buffer (final pH 4.2) (19), and veronal acetate buffer (final pH 4.2)(21).“ NB: Mind the different pH’s of the washing buffer vs. vehicle for UO2-acetate!   

perhaps also of value:

http://www.ou.edu/research/electron/bmz5364/maleate.html;  https://www.researchgate.net/post/how_to_prepare_a_maleic_acid_Tween_20_solution;      

 [ok, used as blocking reagent in IHC, SIGMA protocol / TDS:  http://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/blocking-reagent.html ];

Uranaffin reaction: a new cytochemical technique for the localization of adenine nucleotides in organelles storing biogenic amines. J G Richards, M Da Prada, 1977 [ https://www.ncbi.nlm.nih.gov/pubmed/144762, or, better: http://journals.sagepub.com/doi/pdf/10.1177/25.12.144762 ];

cf. also: Chem. reactions:  „ancient“ article (but good to know) by

MORGAN E.J., & FRIEDMAN E., 1938: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1264098/pdf/biochemj01038-0099.pdf:        Citation of SUMMARY: "Maleic acid reacts with thiol compounds with the formation of addition compounds. In the case of thiolacetic acid and glutathione, maleic acid  is [isomerization]  rearranged  simultaneously to fumaric acid." 

Dear Wolfgang,

Thank you very much for your detailed response and valuable included resources.

I remembered maleate buffer with uranyl acetate, but didn´t recall the necessity to get rid of phosphate ions, as I never used phosphate buffer, relying on cacodylate or HEPES buffer, which never caused my trouble.

My recipe for HEPES buffer is as follows:

We store the solid acid HHEPES (a white powder), from which we produce 1M-stock solution by dissolving the appropriate amount in a little less than the final amount of Aqua bidest. The solution is quite acidic, and the pH is set by adding NaOH, which forms the actual Na-HEPES / HHEPES - buffer system. Because of this, one needs a rather large amount of NaOH, so I usually start with strong solutions, switching to less concentrated ones when I approach the desired pH value (e.g. 7,2 for many applications, especially mammals, 7,56 for insects, 7,8 for oribatid mites). Finally, I add Aqua bidest to reach the final volume, check the pH again and adjust as necessary.

One problem is, that HEPES buffer has a high temperature dependence, compared to other buffers, so it is advisable to prepare the buffer at the temperature it is to be used.

To get an idea of the required amounts, volumes and temperature effect, it is worth checking this very nice online tool beforehand:

http://www.biomol.net/en/tools/buffercalculator.htm

or, alternatively:

http://www.currentprotocols.com/WileyCDA/CurPro3Tool/toolId-3.html

For use, I dilute 1 part of stock solution with 9 parts of Aqua bidest to get 0,1M Hepes buffer.

In preparing fixating agents, I usually prepare 0,2M HEPES buffer of half the volume I need for fixation, add the aldehyde solutions and what else is needed (e.g. saccharose to set osmolarity, electrolytes like magnesium sulfate, ect.), and fill up with Aqua bidest.

For insects and oribatids, I also got satisfactory results using 0,05M HEPES pH7,56 (or 7,8, respectively) in the working fixative (1.33% formaldehyde, depolymerised from paraformaldehyde, 1.66% glutardialdehyde, with an addition of 4% (by weight) saccharose, and 6,6µM magnesium sulfate.

The washing buffer I prepare from the stock solution by adding water as described above. It measures pH 7,2 (for mammals/ most cases) at 0°C, 0.1M molarity, and 102 mOsmol as determined by freezing point.

For osmification, I usually dilute the 4% stock solution of osmium tetroxide with Aqua bidest to 2%, and add the same volume of 0,2M HEPES at the desired pH.

best regards

Paavo

http://www.biomol.net/en/tools/buffercalculator.htm

http://www.currentprotocols.com/WileyCDA/CurPro3Tool/toolId-3.html

Dear Paavo:  

many and cordial thanks for this your really scientific protocol which - after a first thorough reading - nothing leaves obscur.  That IS really a straightforward practical instruction one (also a novice!)  can follow easily. I have seen some Materials and Methods sections mentioning the use of HEPES (and eventually PIPES), its / their molarity and the pH value (either overall for the fixative or the buffer per se) but NOT how to really produce the stock and working solutions (saying something also about change of pH with altering temperature, etc.)   And, as a side effect, one can see that the "nucleus" of the protocol is not  "make AA, BA  and dilute / mix AA and BA and swirl counterclockwise..." (:-))  If I am allowed I shall add this your Protocol into my e-Working Protocol Library.  Thank you again and take care! Best regards and wishes Wolfgang

Dear colleagues, i just discovered this page, which gives a wealth of information regarding HEPES and its applications, including several recipies for the preparation of stock solutions:

Best regards to all, Paavo

http://www4.mpbio.com/ecom/docs/proddata.nsf/(webtds2)/15884

As the rutine technique,I have prepared cacodylate buffer with 7.5% sucrose and used for glutaraldehyde  fixative and  osmium fixative as well as for washing.

I have applied cacodylate buffer with sucrose, for glutar aldehyde, wash and osmium. There are no problems. Details have describedmy monograf in Forum for Nordic dermatovenereplogy, 2010. Nr 3,vol。21．

Kobayasi

Dear Prof. Dr. Takasi Kobayashi!

Ignoring for a moment that you already for some years now are retired from your active position I nevertheless would express my heartfelt reverence for your scientific work in your long lasting active position as leader of the ultrastructural dermatohistopathology branch at the Bisbebjerg Hospital, Copenhagen University.

Regarding your hint „details have been described in my monograf in Forum for Nordic dermatovenereplogy 2010, No 3 vol.21“ :

For the sake of correct bibliographic data and personally as well as now also publicly acknowledging your huge efforts in diagnosis and morphology issues of (human) skin ultrastructure, I honestly would like to add the following:

„The Dermis – Ultrastructural Histopathology of Connective Tissue“

By Takasi Kobayasi in: Forum for Nord Derm Ven Vol.21, issue 3, 1-168, 2016

https://www.medicaljournals.se/forum/?page=issue&volume=21&issue=3

PDF (OPEN ACCESS): @https://www.medicaljournals.se/forum/articles/21/3/1-168.pdf

Forum for Nord Derm Ven (Forum for Nordic Dermato-Venereology) is the Official journal of the Nordic Dermatology Association, ISSN 1402-2915)

[Forum for Nordic Dermato-Venereology - Medicaljournals.se

(https://www.medicaljournals.se/forum/) (https://www.medicaljournals.se/)

In order to provide young students (but also readers and lecturers) in Medicine, Pathology, Dermatology, Biology and other research fields might find this your excellent monograph on the Connective Tissue in (human) dermis more easily, I strongly recommend your uploading the article‘s pdf into the CONTRIBUTIONS section [https://www.researchgate.net/profile/Takasi_Kobayasi/contributions] of your Personal PROFILE in Research Gate *) (https://www.researchgate.net/profile/Takasi_Kobayasi) where in the „Project“-section“ you can find a comment I added already Sep 23, 2016:

https://www.researchgate.net/project/Ultrastructural-histopathology-of-dermis

Collegial and cordial greetings from „retiree to retiree“,

yours respectfully,

Wolfgang Muss

*) naturally only possible if the Publisher permits such action.

Dear Dr Muss,

Kindly tell me your correct　mail address.. I have once sent a reprint of my article "Dermis in Forum" but the mail return to me.

We should spend time with electron microscope. 　　ｋｏｂａｙａｓｉ

Dear Prof. Dr. Kobayasi, thank you so much for your kind reply as well as your attempt to send me the requested pdf (which is approx. 7.200 kB in size). For whatever reason this your message with the attached pdf did not get through regarding the transfer by e-mail to [email protected]: I thank you once again for your efforts.... Due to the fact that fortunately your important contribution was designed to be published 'Open Access=OA' I already have got my personal copy into myprivate e-library. Thank you!

Regarding your friendly invitation to get back to roots (spend time with EM..) I just have to tell you that the EM Lab I run in my active profession at the Pathology Institute has been closed and dismantled (due to financial reasons, I guess) and 35 years of knowledge and know-how have been wasted from one day to the other without any discussion with / by my former employer....(;-() That's the end of it. Period.

No new time(s) approaching... I gladly would get back to the "machine", also if it were only looking into ultrathin sections of special cases... cordially yours, Wolfgang