Hello everyone,
I am working with Precision Cut liver slices of 250 µm. These need to be processed for FFPE. As you can imagenie they are very fragile and tend to break when I embedd them in Paraffin after fixation and dehydration.
Does someone have experience embedding thi tissues in HistoGel before Processing? My Plan is to fix the slices in Formalin over night, then embedding them in Histogel followed by processing for FFPE. Would it make sense to fix the HistoGel, too?
How does HistoGel behave when cutting?
Thanks in advance :)
Best, Franziska
I've not used Histogel for supporting thin tissues, but I think that is not a bad idea. Our lab has used it previously for embedding of organoids with good success. You would want to perform fixation after embedding in Histogel.
However, I think part of your problem may come from overfixation. Overnight incubation in 10% formalin (4% formaldehyde) is sufficient to fix entire (mouse/rat) organs.
Dear John Hardy Lockhart,
Thanks for your reply. I also found a nice protocol on using HistoGel and will try that.
We do fix the Tissue slices at 4°C over night. But after fixation the tissues are not that brittle yet. IHC is still possible, so I don't think it's completely overfixed. But I will try to fix shorter.
We routinely fix and paraffin embed 300um tissue slices with standard methods, and don't have issues with the tissue breaking. You don't mention when the breaking occurs, but if it's when you're doing your sections, it suggests that your tissue is too dehydrated, which can make it brittle. Long fixation in standard formalin (ie. 4% formaldehyde) does not make tissue brittle, it's the processing that does. I would check your processing schedule, and discuss with experienced histotechnologists whether it should be adjusted. Processing schedules made for standard tissue samples might be too long for these small tissues.
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