I am having issues with a histology technique. I'm currently cryosectioning sciatic and L4 spinal nerves, followed by staining with toluidine blue. My section are coming out nicely but quickly degrading within a minute. Is this due to OCT compound? The tissue was fixed in 4% paraformaldehyde and stored in 20% sucrose. I am attempting to count the quantity of fibers in the tissue samples. Thanks!
Ryan, would you mind to clearly define what you mean with "quickly degrading within a minute." (since 'sections are coming out nicely..') You are "cryosectioning sciatic and L4 spinal nerves" of WHICH species? (size?) ...If you meant a kind of "dissociation of cross (?) or longitudinal(?) sections right after thawing of sections unto the (RT-warm) slide: this could be a consequence of inadequate connective tissue properties within and around nerve bundles/ individual nerve fascicles. Since you fix/fixed the nerve tissue already, IMHO, reacting the material additionally with OsO4 and processing with PPD (para-phenylenediamine) in the 70% EtOH-step, dehydration as usual and following embedding in paraffin could be an (a better) alternative for reliably counting of fibers.....(there exist also methods using DAB to stain myelin sheaths....) Not knowing about the task you have to achieve....but just pointing to the possibility of (e.g.epoxy) resin embedding of tissue fixed, treated, processed by standardized methods...(cf. attached image). Best wishes and good luck!
Here are a couple pictures. These are different sections but it demonstrates what I mean by degrading. A soon as the tissue sections come in contact with solution (stain), the structural morphology of the axons appear to dissolve quickly. I think switching to paraffin embedding might be the best option with the addition of OsO4. These are cross sections of Mus musculus sciatic nerve. Thank you for your suggestions Wolfgang H. Muss.
@Ryan: thank you for posting these images.....my guess(es): either too short (pre-)fixation of nerves [try to pour fixative in the moment you can see first the prepared nerve(s)], or poor adhesion of (frozen-) thawed sections on slide(s) or too aggressive staining (i) handling or ii) staining solution and or washing. Therefore my question: would you mind also to give us detail for the Tol-blue staining (mixture of solution, application mode). Thank you!
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Histological Techniques