Have tried multiple times in 70% ethanol, 2 washes in 95% ethanol, three washes in 100% ethanol, 2 washes in xylene, and three separate washes in paraffin. Tissue has been fixed for over a year in formaldehyde
Standard formaldehyde solutions contain methanol that can extract lipid Histopathology
Volume 41, Issue 1, pages 75–79, July 2002
Thank you Michael. I was wondering if you knew a standard protocol for processing fresh fat tissue. I have tried looking online and I am not having very much luck. Thanks again!
Try the above article. Alternatively, use alcohol free formaldehyde from EM (5ml aliquots under argon) and process as frozen section to retain fat. Some clinical labs use microwave fixation in formalin. Here is new technology: Ultrasound-accelerated formalin fixation of tissue improves ...
http://www.nature.com/modpathol/journal/v18/n6/full/3800354a.html
Ultrasound-accelerated formalin fixation of tissue improves morphology, ... Fat-abundant breast tissue is difficult to fix by conventional methods.
Do I understand correctly - that your tissue has been already fixed for over a year in formaldehyde?
Michael's advice is for a prospective study - do you plan one? The ultrasound assisted formalin fixation is around from early 1990s, but not very popular with lab equipment manufacturers. For microwave assisted fixation you need a special lab microwave with temperature control – quite a few on the market and most of the labs have them. I have used this method for research samples and it is a very good and reliable.
If you want to work on those specimens which are still in formalin after a year - you do not have much choice. In such case taking samples (3-4 mm slices) of your area(s) of interest and very thorough washing in buffer (PBS pH 7.6, easiest way is to make the buffer from Sigma tablets 1 tablet/200 ml) would be a start. Very old pathology manuals allow up to 5 days washing in buffer, but if you have relatively thin slices - I would suggest 24 hours, at least 4 changes of buffer, on rotator, at room temperature. You didn't specify what staining methods you want to use - if histochemical staining - you might need to modify the methods (as over-fixation diminishes stainability. If you hope for immunohistochemistry - that indeed would be a challenge. I have used immunohistochemistry on archival over-fixed tissues and on very old mummified material and it is possible to get results after experimentation with different antigen retrieval methods and avoiding false positive/negative results using positive and negative controls.
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