I'm an undergraduate working on a project where I have intestines that were fixed in formalin before I arrived. They have remained in 10% neutral buffered formalin this whole time. I am embedding in paraffin and cutting at 5 micrometers. Many of the samples vary in color and fragility from one to another. Some are white, some are dark brown, some are a translucent pink. I'm also noticing some strange issues in my stained slides. Examples: no villi present, just a solid mass of something stained slightly blue with dark blue clusters of material on the slide; what look like goblet cells all clustered in a semicircle at the end of where villi should be; obvious gaps between muscle cells in the muscularis externa. I don't think it's my staining procedure as it worked beautifully on another sample of the exact same species and tissue type. I'm starting to wonder if some of the samples were not stored correctly or quickly enough.
Hi Gregory,
2 years is a long time for fixation. Especially since the fixation is a continuous process. Over-fixation may lead to tissues that easily break.
The differences in colour may be due to the amount ofred blood cells in the tissue.
You can also get all kinds of artifacts when dehydration is not performed correctly.
With kind regards,
Ronald
I think it would be ok. Maybe prolonged hematoxylin staining is necessary. Long storage in formalin, especially in not-buffered formalin, gets a lower pH due to formed formic acid. That leads to chromatin break down. On the other hand formaldehyde is bound to cytoplasmic proteins and "demasks" binding sites for eosin. So a fainter eosin staineng may follow prolonged fixation.
Hi Gregory,
to store a tissue 2 years in 10% formalin appears far to long. We had a similiar observation on prmate tissue stored for 5 years in formalin and we got irregular staining and all kind of artefacts. Also the tissue becomes quite hard and difficult to section. I would not recommend to use it. Usually, after proper fixation, tissues should be immediately embedded in paraffin, where it can be stored quite a long time.
Regards, Fred
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