Souravi Sinha,

it would be of great help if you could state your question more precisely: i.e. skin animal, skin human, location, certain needles (which one?), has your tissue already been fixed (and if so, data of fixative, etc.etc.). You would like to omit sodium cacodylate (as a buffer solution, I guess) and OsO4 as well, for what reason, which background?  From experience I know there are some papers out in the wild. It makes no sense to give any specific and valuable advice not knowing some detail of your work.

The skin tissue to be analysed is rat ear skin. The usual procedure for SEM tissue preparation requires Osmium Tetrachloride which is not easily available in a lab. Hence I want to know if it can be substituted with a simpler compound.

Dear Souravi,

....„requires Osmiumtetroxide/Osmium-tetra-oxide“....if you can’t /will not use that stuff this might hamper (but not necessarily, depending on your 'analysis') your preparation results …

to post here most rapidly for your convenience, see what I have written in another, former RG-thread ( cf.: https://www.researchgate.net/post/Is_there_any_OsO4_osmium_tetroxide_alternative_for_fixation_in_immunogold_labeling_for_TEM?) you could try (see below):

Since there have been some earlier question-threads in ResearchGate regarding this matter, I would like to point you to them for reading (and perhaps get answers too):

https://www.researchgate.net/post/Chemical_fixation_for_TEM_samples-Os_alternative

(find there also the answer of Alba Jimeno Romero,Re to me and Vladimir Dusevich, but this is meant for TEM-preps )

At:

[http://cellbio.med.harvard.edu/research/research-facilities/conventional-electron-microscopy ] attention:  meanwhile this URL has been changed ! to:

you will find an interesting information, [which will solve your problem with “invisibility of specimens after embedding into resin”, not applicable for SEM]:

Osmium Free Method [for Resin embedding] (Tannic Acid / Uranyl Acetate method, NB added: this method without embedding into resin perhaps could be perhaps used too as a primary processing until 70% EtOH, then proceed as usual for SEM)

1. Fix in PFA/ Picric acid as above. (NB: this fixative you’ll find on the webpage too)

Do the following processing on ice until NB added: 70% // 100% Ethanol:

2. Freshly made 1% Tannic Acid in Maleate Buffer (MB) for 40 min, then rinse 2x in MB

3. In the dark: 1% Uranyl Acetate in MB for 40 min, then rinse twice in MB

4. Dehydrate 5 min each: 50%/70% Ethanol

5. 1% PPD(p-phenylenediamine) in 70% Ethanol for 15 min *)

6. Rinse twice in 70% Ethanol

7. Dehydrate 5 min each: 80%/95%/2x100% Ethanol

8. Resin infiltration (LR white or other hydrophilic resin)

9. Polymerization at 40° C

*) p-phenylenediamine will your tissue (no problems in terms of retained antigenicity) with a brown to dark-brownish-black coloration.

Since you asked also for a substitute of sodium cacodylate (guessing either you cannot order it or don’t have the source for it or don’t want to use it because it contains As=Arsene and therefore is considered to be a hazardous component) I only can point you to use “normal, physiologically 'unhesitating' buffers” like Na-Na-PO4 (sodium monophosphate-sodium diphosphate at the right pH and m-osmolarity) buffer or even PBS (cf. Vladimir Dusevich's answer to ‘ Is cacodylate buffer necessary in SEM protocol, or I can remove it?‘  at:  https://www.researchgate.net/post/Is_cacodylate_buffer_necessary_in_SEM_protocol_or_I_can_remove_it? :

< I second that suggestion. PBS is a good substitute for cacodylate buffer for SEM >

Best wishes for the progress of your work,

regards, Wolfgang

I welcome  explanations for working with EM from researcher Wolfgang H. Muss. I can send a book chapter also wirh protocols for EM.

Regards

Carmen