Frozen Section Formalin fixed human tissue

[justforgotfrag]

Hi All. my research focuses on imaging analysis, but then before that, I need to get a clean slice of tissue. So far, it has been unsuccessful and I was hoping that someone here can help me out.

I am currently using Leica CM 3500 XP. my formalin fixed human block is embedded in 4% CMC. the temperature of the crytome is at 20C and the angle of the blade is 20. our goal is trying to get <50 micron. the setup seems to be fine but then when we tried to slice it, the tissue did not stick to the pink tape, as it would just fall off. I was wondering if anyone here familiar with this type of slicing..

Thanks!

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[deleted314957]

I've been doing frozens for more than 30 years and never heard of "pink tape" and if you want decent microscopy on a frozen section consider about 5 microns.

 

[Autopsy101]

I don't know what pink tape is either...However, when I have cut a frozen on formalin fixed tissue and not fresh - there usually are problems getting the tissue to adhere to the slide throught the entire staining process without floating off. We use positively charged or plus slides when we cut them on a formalin fixed specimen to try to minimize that issue.

 

[TMZ2007]

Forgive my ignorance, but what would be the reason for doing a frozen section on fixed tissue? (As opposed to just processing it routinely and embedding in wax?)

 

[lipomas]

Why would you need to freeze it? You can cut a section off of a paraffin block - that's the entire purpose of the paraffin block. Freezing it makes no sense.

 

[KCShaw]

You need someone on-site who knows what they're doing, or send it out.

One can certainly do a frozen on formalin fixed tissue, although I agree there needs to be a good reason to do a frozen section rather than embed in paraffin.

 

[reviliver]

When we do frozens on fixed tissue in the GI path research lab where I work, we soak it overnight in 6M sucrose before freezing which helps it freeze better and adhere to the plus slides. Definitely the plus slides are important for getting the tissue to stick. We also do closer to 5 microns; I think usually about 7.

Not sure if any of this is useful to you and I'm just a tech, so take with a grain of salt.

 

[horrendoma]

Forgive my ignorance, but what would be the reason for doing a frozen section on fixed tissue? (As opposed to just processing it routinely and embedding in wax?)

Maybe he's trying to do some lipid stains on archival wet specimens.

 

[Torsed]

I do my image analysis on my scope in about 30 seconds without a high tech computer or software.

 

[skatalite]

When we do frozens on fixed tissue in the GI path research lab where I work, we soak it overnight in 6M sucrose before freezing which helps it freeze better and adhere to the plus slides. Definitely the plus slides are important for getting the tissue to stick. We also do closer to 5 microns; I think usually about 7.

Not sure if any of this is useful to you and I'm just a tech, so take with a grain of salt.

I've been working in a research pathology lab for over a year. This person is correct. You don't generally fix frozen's with formalin, you add 30% sucrose. That may be the reason you are getting crummy slices. Also, fresh frozen...no sucrose obviously, frozen with liquid nitrogen should slice well, but I do know that every tissue requires a different temperature. In fact if your having issues getting a clean slice with fresh frozen tissue, you can press your thumb to the block to warm it up. Don't keep the cryostat door open to warm up because that will add humidity. Also, the comment earlier about the positive coated slides are very important for immuno or any kind of high heating step. Thermo's superfrost positive coated slides are great or bone-rite slides are great too. If you don't need the slides for immuno...mainly flourescence, you should just formalin fix and paraffin embed...much better morphology than frozen.