I wish to do the histological analysis of my silk sponges. For that I need to slice my scaffold through microtome but I am not getting the results. My protocol is like i put the scaffold in 4% formalin fro 24 hours, then pass through ethanol grades to dehydrate, transfer to xylene and embed in paraffin. But when i slice it through microtome so it slice the paraffin part but is unable to slice the scaffold part. Kindly help me out that what's the issue and what i am missing? Picture of the sliced scaffold is attached where the mid round unsliced area is the scaffold.
The above pics are of cryostat but the microtome is giving the same results, is this the issue of treating with xylene?
Hello Numan,
Please let me know your protocol in details.
For example, how long the material stays in each solution?
Another question. In the composition of sponge is there any kind of mineral element?
well if we come to the material fabrication so i followed the protocol of Rockwood 2011, i extracted silk fibroin, hydrated, lyophilized and added HFIP and salt leached. I don't know if salt has an issue as i have kept it in water for 3 days and made sure that there is no traces of salt left. and for paraffin molds i kept in 4% formaldehyde, then dehydrated with 70%, 80%, 90% and finally pure ethanol. I kept them for 20 minutes each in ethanol grades, then i shifted it to xylene for 4 minutes covered if with aluminum foil. Finally i made the mold by putting the scaffold in mold and poured the wax inside. now when it comes to slicing so i have the issue i questioned above, Dont know what to do, My PhD work relies on this and i am not sure what to do with it. I need little guidance. Your help will be really appreciated :-)
ok.Your material is inside of a histological cassette? If your answer is yes. You must leave at least 50 minutes in 90% ethanol and more 3 times of 50 minutes each in pure ethanol (absolute ethanol P.A.). After this, you must put your cassettes in xylene (absolute = pure) at the same time (50 minutes) in 3 differents xylenes (named I, II and III respectively) as you did with the ethanol. Then, you must put you material inside the hot wax (65 Celsius degrees ) in 3 differents pure wax (named I,II and III), for 60 minutes each. Only after this steps you "block" the material. Before slice you must put the block in the refrigerator for 50 minutes, to cool the wax.
Your material is with holes because you didn't left the time enough in each solution.
Try this protocol, I think that will work. But if there are salts or sand within your material you will not be able to slice. You have to be sure that all salt/ sand was removed before.
Thanks Alessandre Hataka for your precious suggestion. I will try to work on this and hope that I get the desired sections.
Okay. I think it will work. Please give me a feedback on this.
Sincerely, Alessandre Hataka
I have sent you texts there on Skype. I guess you are offline or don't know why you didn't Recieve them so far
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