I am working on termites and doing in situ hybridization but at the end, I can't see cells in specimen slides. my question is how to maintain the structural integrity during fixation or whole process can influence cell structure. I think fixation is the main part of hybridization. I am using 4% paraformaldehyde at 4degree to fix termite for 3-4 hours then gradient dehydration with ethanol. if any suggestions it would be highly appreciated.
Maintaining the structural integrity of cells during fixation is crucial for preserving their morphology and cellular components for subsequent analysis. Here are some key tips for achieving successful fixation while preserving cell structure:
Noor Us Saba,
for any colleague reading your request it would be fine, really informative and therefore also important for a decision, how one can help with regard to your problems if you would add at least 'little' additional information about your performing in-situ-hybridization and adding - perhaps and if available - (an) image(s) of the final appearance on slides...
Further (always informative and sometimes recommended) readings (out of many more);
ZHU et al, 2021: Effects of fixation on bacterial cellular dimensions and integrity [iScience. 2021 Apr 23; 24(4): 102348] cf. also:
Article Effects of fixation on bacterial cellular dimensions and integrity
(=https__://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066382/ NB: prior to 'execution' - when copying that URL into your browser - delete the two undersigns__ in between http and ://www....) or directly on NIH-NCBI-PubMed-website (4,6 MB) :https__://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066382/pdf/main.pdf
(NB as before);
Biotechne(R)
What is the Significance of Fixation: cf.
https://www.bio-techne.com/imaging/immunocytochemistry/fixation-and-permeabilization-in-icc-if
ETHOS BioSciences:
Choosing the Right Fixative
https://www.ethosbiosciences.com/choosing-the-right-fixative,
Steps to Better ISH and more... by Geoffrey Rolls, BAppSc, FAIMS, cf.:
https://www.leicabiosystems.com/de-at/knowledge-pathway/steps-to-better-ish/
Best wishes and regards, W.H.M.
One of the conditions for the preservation cell structures is mainteinance of the osmaticity of solutions. In case of the prefixation with OsO4 the osmolarity of the solution is adjusted with sucrose for 400 mosm(add 25mg of sucrose is added in 1 ml solution of 1% OsO4)
Wolfgang H. Muss I am adding one of my Silde Zoom view though all of your information is valuable I tried a lot but could not get suitable results. i will check your shared article and other information about the significance of fixation. my gene of interest are present in cuticle.
Kais Khudhair al Hadrawi yes i am using 4%paraformaldehyde paraformaldehyde for the first time I used ti fix it overnight but did not get any results after that I used a 3-4 hour fixation at room temperature of 24 degrees and gradient series of ethanol, my slide is not so much clear when I see it at low magnification; it seems good but at high magnification some time I even can't see. this time I will do the post-stabilization, method as you mentioned. thank you for your kind response
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