I am using this protocol (https://www.scientistsolutions.com/forum/cytology-histology-and-ihc-organelle-and-cytoskeletal-staining/cartilage-staining) to do cartilage staining on organoids that have been fixed in 4% PFA, dehydrated in 10,20,30% sucrose+sodium azide, frozen in tissue freezing medium, sectioned on a cryostat and kept at -30*.
Would I need to take any additional steps before re-hydrating the sections and next staining with the hematoxylin?
Thank you in advance!
If they are frozen, you can fix in 4%PFA for 30 minutes than wash 2 times with PBS (no special hydration steps) and go ahead with either staining
or
if they are paraffin embedded tissue sections than you need to deparaffinize and hydrate as
I have protocol for paraffin as below;
Safranin O staining: Paraffin sections were deparaffinized in xylene (3x, 3min), dehydrated by treating with decreasing concentrations of ethanol [(100%, 95%, 70%, 0% (dH2O), 2x, 3min each)] and finally placed in dH2O. The sections were stained in Weigert’s iron hematoxylin (5min), rinsed in changes of dH2O till leaching of blue coloration stopped. Sections were differentiated in 1% acid-alcohol (2secs), rinsed in dH2O (3x), treated with 0.02% Fast green (1min), 1% acetic acid (30secs) and 1% Safranin O (10 min). The sections were dehydrated in 95% ethanol (3x, 3min), 100% ethanol (2x, 2min), cleared in xylene (3x, 3min) and mounted in Permount™ mounting medium (17986, EM sciences). Proteoglycans stain red, cytoplasm stains gray green and nuclei stain black.
I think you need to fix fresh frozen sections in 10% formalin prior to going for staining procedure (especially H&E). I use to this for amphibian gonadal tissue for better clarity and photography purpose.
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