Dear Tim, please define your tissue to be examined and - if possible - also, if you have in mind already some specific method for demonstrating / localizing adrenergic/cholinergic substrate...just to know where to begin...(:-)) best regards, Wolfgang

I examine the ganglia located in the cardiopulmonary junction (most proximal centimeter of the pulmonary veins. After a treatment with KMnO4 i should be able to see differences between the adrenergic and cholinergic synaptic vesicles. But the problem is how to sample such a small ganglion to have a tissue bloc small enough for electromicroscopy?

Dear Tim, guessing your spec's are from human? (or animal? which one)?,

knowing this perhaps leading to some hard work... not knowing if there is a possibility to try imaging the “true” location of the ganglia by e.g. MRT or NMR or other imaging techniques:

it might be practical (if you don't have a precisely defined anatomical area where the (“your”) ganglia are, don’t you? ) to

i) choose a suitable resection margin at the cardiopulmoray junction, set this as zero 0-point, process either for cryo- or paraffin techniques: Start with (e.g. 2-5 µm serial ) cryo- or paraffinsections + evtl. immunostains for ganglia/neurons, reconstruction from sections – at least 2D of dimensions of the sectioned area/volume.

ii) define the “most probable location” - of the ganglia you would like to see - by kind of stereological principles / measurements for the tissue you are working with.

iii a) then try to transfer the previously found to "fresh tissue", take out randomly 2-5 slices [ say each 1 mm thick, but see also iii b) below], fix as appropriate for your task and process/embed for semithin section / TEM-observations.

The following methods applying to blocks of samples embedded “oriented” in your resin:

iii b) or prepare at least parts of the cardiopulmonary junction / pulmonary veins as a "tube"./ or "half tube".. having a defined “0 (zero)-point” [e.g. point of resection from the ultimate angle/corner of the cardiopulmonary junction = zero 0µm] and an “end zone” (= zµm), the whole dimension / length or height of which will be (by your definition = xy µm) divided – say - into 3 zonal segments/sectors.(shrinkage factors due to fixation as well as processing/sectioning have to be calculated).

From each of those defined segment zones you only will section 2 or 3 semithin sections randomly (see iv)

iv) semithin sections (1-2 µm thickness), either serial or following sterological principles (e.g. choosing sections randomly in those 3 defined sectors, e.g. always within a defined distance in the range of say 25 to 50 µm - this depending on the dimension/volume of the ganglion/ganglia you want to hit. Naturally there will be a problem if your ganglion/ganglia is/are only 25 µm in dimension!).

v) following this principle you will find a probabilistic value, where your ganglia "normally" are situated...

vi) if those values are established (either from serial section reconstruction or from the stereological approach), you should be able to macroscopically prepare "blindly" your specimens under a dissecting microscope – stereomicroscope using the distance values found previously, expecting at least some hits in one or two of your multiple semithin sections (and the respective block then can be trimmed for ultrathin sectioning).

By the way: would you like to share information about the KMnO4-fixation of your specimens?

Best regards, Wolfgang

Thanks for the explanation, i keep on trying to book some progress.If i have some results concerning the KMnO4 fixation i will let you know because i don't know yet if it has to be combined with other fixatives or used as a post-fixation step.

regards, Tim

Dear Tim, thank you for your reply.

KMnO4-fixation in EM-specimen preparation procedures generally seems to be treated just like as an orphan …

There for sure exist some special recipes… which cannot be found easily – most “EM-(hand-)books” [e.g. HAYAT, BOZZOLA, MAUNSBACH, and others] have included a really small section and on that matter.

There are some (older) methods published indicating the use of only hydrous K(or Na-)MnO4-solution as the initial fixative. The preservation of ultrastructural morphology will be limited to specific structures or tissular compartments, however. With do you mean kind of "staining" ultrathin sections? or immersion/incubation of specimens as a whole after (PFA)/GA-fixation substituting the OsO4-immersion?

Hopefully not bothering you with an additional longer reply (not to demonstrate my poor practical knowledge in KMnO4-fixation but only to be perhaps of help in finding answers to your interesting practical problem!) I would like to point you to the following:

Find the ResearchGate-thread: https://www.researchgate.net/post/Chemical_fixation_for_TEM_samples-Os_alternative

for an interesting hint on KMnO4-Fixation: Find the answer of Yaroslav Tsytsyura (including an EM-Image) in this thread. He says:

As a really old reference (you might have found meanwhile by yourself) see:

J Biophys Biochem Cytol. 1961 Mar;9:711-6.

WETZEL BK.

PMID: 13784665 [PubMed - indexed for MEDLINE] PMCID: PMC2225023 Free PMC Article @ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225023/ or

or: http://jcb.rupress.org/content/9/3/711.long

or: JOHNSON RPC:

in: Planta(Berlin) 68, 36-43 (1966) (NB: This article is not listed or cannot be found in PubMed) but – if you would like to read the article (with perhaps interesting considerations on reaction mechanism in the view of our predecessors) I could send to you a self-scanned pdf.

Search in PubMed/NCBI for: < permanganate fixation AND Ultrastruct* > yields 81 references,

Search in PubMed/NCBI for: < Potassium permanganate fixation AND Ultrastruct*> yields 59 references,

Search in PubMed/NCBI for: < Potassium permanganate fixation AND EM > yields only 3 references, one of them is PMC free article:

Issidorides MR, Havaki S, Chrysanthou-Piterou M, Arvanitis DL.

Ultrastruct Pathol. 2000 Nov-Dec;24(6):399-405. PMID: 11206337

Song J, Lee C, Lin CH, Chen LB.

J Struct Biol. 1991 Oct;107(2):106-15. PMID: 1807347

Doggenweiler CF, Heuser JE.

J Cell Biol. 1967 Aug;34(2):407-20. PMID: 4166578 @ http://www.ncbi.nlm.nih.gov/pubmed/4166578;

Free PMC Article @ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2107310/

Good luck and best wishes,

Wolfgang

Dear, thank you for the detailed responses but the fixation with KMnO4 didn't work as good as expected. But on the other hand, it was possible to search for ganglia in the semithin sections after making the blocks based on the most logical place of occurence, this is in this case where muscle, connective tissue and fat lying close together. I send you also the article in which the use of KMnO4 is indicated to create a certain density to distinguish adrenergic nerve terminals from cholinergic ones.

greetings

Tim

Dear Tim,

I am glad you ended up at least with finding the ganglia you were looking for.

Longer ago I had to find ganglia in the Auerbach's plexus (GI-Tract) in semithin sections too (for alterations = 'lysosomal storage" of lipidic material due to NCL = neuronal ceroid-lipofuscinosis in patients, cf. https://www.researchgate.net/publication/200468964_Electron_Microscopy_as_an_important_diagnostic_tool_in_Neuronal_Ceroid-Lipofuscinosis_%28NCL_Batten%27s_Disease%29?ev=prf_pub )

Thank you for remembering your intention to let me know progress of your work so far after such long time.

Meanwhile I learnt (from further 'old' literature refs that the use of KMnO4-(fixation/incubation) for localizing NSG or differentiation of adrenergic vs. non-adrenergic neurons might be trickier than previously thought. Nevertheless an interesting and intriguing task.

Thank you again for sharing your result (if you would like to discuss further please just send an e-mail to my account at work, [email protected]).

Best wishes and good luck, Wolfgang

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