In order to utilize SEM (Scanning Electron Microscopy) technology, tissue samples typically undergo a fixation and then dehydration process. While creating the protocol for my thesis research, I have been trying to explore if there is any research for what these minimum or maximum time intervals are for each step during a graded series of ethanol, especially what the supporting research/evidence is.
I have found some typical protocols, but no luck in finding studies, equations, or reasoning that explain why the specified time intervals were utilized. For further context, I am creating a protocol for bovine and porcine tissue samples.
Any ideas, suggestions or recommendations would be incredibly appreciated! Thank you in advance for any help or assistance.
Dear friend Tarnvir Dhaliwal
I found some information that might be helpful. Ethanol is miscible with water in all proportions so that the water in the specimen is progressively replaced by the alcohol. A series of increasing concentrations is used to avoid excessive distortion of the tissue. A typical dehydration sequence for specimens not more than 4mm thick would be: 70% ethanol 15 min; 90% ethanol 15 min; 95% ethanol 15 min; 100% ethanol 15 min; 100% ethanol 15 min; xylene substitute (2 changes) 10 min each (Can you leave fixed tissues in 70% ethanol for a prolonged time before ....).
I also found a post on ResearchGate where a user asked if fixed tissues can be left in 70% ethanol for a prolonged time before finishing the embedding protocol in paraffin. One of the answers was that generally, 24-48h storage in 70% ethanol at 4 degrees should not have negative impact on most of the tissues in most of the staining routines. For paraffin work, stopping steps at 50% EtOH and 100% EtOH are normally overnight. Better stopping points would be to fix and then let the tissue sit in fixative (Tissue Processing Overview: Steps & Techniques for Histopathology). After washing, you could store the tissue in 1X PBS or in 70% ethanol. Post fixation, tissues can be maintained in 4 degrees or in room temperature. After fixing the tissue, the next step would be passing them to 60% ethanol. There, you can leave them in 60% ethanol in the fridge at 4*C until the next step of dehydration (higher concentrations of ethanol).
I hope this helps! Let me know if you have any other questions.
Source:
Tissue Processing Overview: Steps & Techniques for Histopathology. https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/.
Specimen Collection and Processing by Collection Site and Biorepository .... https://brd.nci.nih.gov/brd/sop/download-pdf/2684.
Immunohistochemistry fixation protocol | Abcam. https://www.abcam.com/protocols/ihc-fixation-protocol.
The Paraffin Embedding Method II: Protocols | SpringerLink. https://link.springer.com/chapter/10.1007/978-981-16-6498-4_3.
Most of the electron microscopists have their own reasoning for their protocol and stick with it, I being one of them!
Anyway, coming to the point the duration of immersing the samples in different ethanol concentration varies according to the sample size.
*For a sample of say 3mm cube I would start with:
30% ethanol for 5-10 min,
50% " "
70% " 15-20 min
80% " 10-15
90% " "
95% " "
100%. " 20-30 min with three changes.
After this you can use HMDS or CPD for drying.
The above mentioned was passed on to me from previous generation.
* All the durations were only arbitrary.
I hope this helps.
There is no Xylene involved in SEM sample preparation.
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