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Author Topic: Is there a standard protocol for frozen tissue embedding?  (Read 2260 times)

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Offline SRajpara

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Is there a standard protocol for frozen tissue embedding?
« on: July 29, 2015, 07:03:43 PM »
Hi all,

I have been scouring the internet and asking people who are experts in the field about an official protocol for embedding organs which are going to be frozen in OCT and then cut in a cryostat and then stained with IHC or histological stains. I'm very new to this, and I am worried that I have messed up all my samples. Here's the protocol I've been using for my project:

-Once we dissect the organ of interest, we submerge it in 10% formalin
-We let it fix for 72 hours, then move the organ to 15% sucrose solution
-Once the tissue has sunk to the bottom of the vial, we move it to a 30% sucrose solution
-After it has been in this for at least 24 hours, we then embed the tissue in OCT and keep in a -80C freezer

Then we section those tissues whenever it's convenient for us. I'm reading several sources that say one should fix the tissue AFTER sectioning. Does the fact that I'm fixing the tissue before embedding have any effect on my sectioning? I've been experiencing tissue loss every single time I do IHC, and I've changed the antigen retrieval buffer, been very careful on each step, etc. yet I still lose a considerable amount of tissue. We use super frost plus slides, and our sections are 20um thick. We work with adipose tissue, which I know is troublesome, but we also use kidney and skin samples, which also fall off the slide often. Please let me know what I can do to fix this situation. Thank you for your help.

-SR

Is there a standard protocol for frozen tissue embedding?
« on: July 29, 2015, 07:03:43 PM »