It looks like problems with slicing...

What device do you use for sectioning? Is it everything ok with its knife?

It can also be a problem with the media you embed your material  which come out too soft for obtaining appropriate sections.

Unfortunately I don't know specific points,  and I can help just by general view

I think you have to mention some details about your processing as I'm sure that the problem is in the processing

Thank you Maria and Hesham.

Maria: We use a Leica ultramicrotome (EM UC6) for cut the thins and semithins from the resin embedded tissue, which is made using an Araldite CY212 kit. Your suggestion that the resin may be too soft makes me think I should use the variant with MNA?

Hesham: as the protocol is very long I have not typed it all out here - please refer to the Nature Protocol in my question for additional details. If you have any other insights into my problem and need more information I will certainly provide it.

The protocol you are using is very specific and I cannot comment on it. However, pictures in second paper (with protocol) are even worse in quality than yours. I believe the protocol was devised not for obtaining overall nice TEM micrographs, but for observation of some specific cells. By the way, gaps in tissue do not look like holes in a section, so I see nothing wrong with cutting (and possibly with embedding).

I think you should vary DDSA. And be sure that its dates determined by the manufacturer did not expired or near to this. Epoxy resins catalysts maybe also sensitive to un appropriate storage conditions which result in incomplete resin hardening.. Even in cases when everything is done right.

Also examine carefully ultramicrotome knifes. Sometimes little chipped pieces or dulls may be a reason of problems you describe.

And the last point on your protocols: I completely agree to Hesham N Mustafa. You should place you protocols directly here and describe in details what exactly and how you act. Yes, all such protocols are long but there are  may be some slight but critical features that are never referred in protocols published in papers. In that case I hope we can help you.

And by the way, don't be afraid to tune published methodology varying different conditions.

I do not understand what is a problem with reading protocol from attached link?

Again, I see no problems with cutting/embedding.

Maria, could you please explain in more detail why you think embedding is a problem?

Dear Vladimir,

I realized that I wrote my answer in a parallel to to you. In the same time.

And here is what I have to say to explain my point of view:

I don't think there is a big troubles with embeddind or cutting. But I think it might be.

we obtained similar pictures if there were  some troubles with resin composition or hardening process. I mean many of haze edges and a big hole in the left bottom corner

Once there were scoundrel times for the scientists in Russia, though we proceeded with our work and as we couldn't recieve some reactives, we had a chance to test our compounds for its ability to keep their features for a long time. And we often faced problems with ultramicrotome seeing similar empty spaces in our sections.

Once I had a problem with inappropriate temperature resulted in very long hardening process or its incompletion.

I agree, that problem may be in the protocol. And that's the reason I want to know exactly all the steps of tissue processing in details to find eventual points to change.  and that' because suggested not to take published protocol as a the only truth. And advised to Katharine Dobson vary different features.

As EM is like an art, isn't it? It always demand very careful aproach in every step to get best results.

Sincerely yours,

Maria

I don't think that slicing is the problem which normally gives other images. It is more likely that the fixation step was not accurate. The time before the tissue was fixated was too long or the penetration of the fixative was not good enough depending on your fixative or sampling method.

Please note: intercellular space is still under discussion, you don't know how large this space is. Second your osmolality of all fluids till your end fixation change the intercellular space. Try to find the Karnovsky technique for electron microscopy or look into Hyat Principles and Techniques of EM Biological applications Vol I Nostrand Reinhold NY ,1970. If possible try to find Lewis and or Shute methods rather old but still effective, because old microscopists had to work with impure substances.

Last: try to find articles of HKP Feirabend  on fixation techniques (I thought in Methods ) Don't have it at hand. Succes and I am interested in your results.

From this only picture is hard to judge, but I think that problem is in fixation or in embedding. I've worked a lot with cerebellar slices for EM . You can try to look into my protocol: :"Morphogenesis and regulation of Bergmann glial processes during Purkinje cell dendritic spine ensheathment and synaptogenesis Glia. 2008 Oct; 56(13): 1463–1477 

How did you perfuse your animal? It may be a perfusion problem. You need to skip PBS perfusion and go right way to Paraformaldehyde. Or you have to reduce PBS prior to PARA. You can see my EM photos are perfect in my papers:

Article Projections of the lateral reticular nucleus to the cochlear...

Article Projections of the Second Cervical Dorsal Root Ganglion to t...

Hi Katharine,

I think those white gaps are normal if you have done physiology on slices before  fixing them.

I believe this is due to the fact that the surface of an acute slice is not perfectly flat (due to the dead cells) and when sectioning you will see parts of your slice popping up 'earlier'. I am sure that if you section deeper in the tissue (>20um) the white gaps will get smaller and smaller and eventually disappear.

If you are interested in the surface of the slice, I think there are few things that can help:

-Use 'vibrocheck' or equivalent if your vibratome has it (Leica VT1200 and the newest Campden Instruments slicer have it)

-Try to optimize the time of your physiological experiment (as short as possible)

-Use or re-calibrate your Microwave-assisted fixation (I can send you a detailed protocol if you need it).

I hope this helps.

All the best,

Vincenzo

Please repeat the processing steps again cautiously.