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Fixation 101: A Primer for Outsourcing Your Histopathology

Clearly, experimental tissue samples are vital to any study.  Before you sacrifice your animal or start preparing buffers, here are a few fundamental techniques and practices that will help you excise an ideal sample before sending those samples to us for further histopathological assessment.

Select the right tools
It’s not possible to do good work with bad tools. If biopsy punches are an appropriate tool for the tissue you’re hoping to analyze, use those. Otherwise, be sure you’re working with straight forceps and sharp scissors. This might sound obvious, but it often happens that we realize we’re working with dull scissors after we’ve begun the procedure. It’s always a good idea to test them beforehand.

Take a little more tissue than you need 
Remember to excise any regions of interest with a bit of extra tissue for manipulation and manual handling, if possible.

Work quickly
Mitochondrial and cellular function doesn’t stop when respiration does. Unless you work quickly, tissues can undergo hypoxic conditions, which can lead to destructive effects on cellular morphology. It’s important to take what you need quickly and get any removed samples into fixative as soon as possible.

Use relatively small pieces.
If you want your immersion fixative to fully penetrate your sample quickly, it’s only logical that small tissue samples are the best choice. If you need to work with large samples, you might consider other methods for fixation besides immersion. We’re happy to consult clients on the best fixation practices for each specific need.

Fewer bigger cuts are better than many small ones
As long as you know exactly where you’re cutting, you’re likely to have smoother tissue sample edges and keep samples intact better when you use fewer cuts. We realize that it’s not possible in all situations and sometimes it’s necessary to make many small cuts. 

Choose your fixative wisely
We generally recommend 10% neutral buffered formalin (NBF) and there should be 10 times the formalin volume to tissue volume.  Depending on your specific needs, however, we can discuss other fixative options with you. 

The duration of fixation is important 
Over-fixation can cause morphological tissue artifacts, especially in delicate tissues like the brain. Under-fixation will, of course, result in an unstable sample that will lose structure and quality with time. We recommend a fixation duration of 24-48 hours when using 10% NBF.

To chill or not to chill - that is the question 
When you fix tissue under cold conditions, you can help prevent anoxic effects from setting in, but at the same time you run the risk of slower diffusion. Some people prefer to use a chilled fix upon initial sample collection, but then allow the fixative to come to room temperature to ensure diffusion coefficients are working in your favor. We don’t recommend keeping samples at room temperature for long-term storage, however, at least until they’ve been processed.

For more suggestions or advice on how to best collect and fix your tissue samples, or if you have any concerns about the protocol you’re currently using, please feel free to contact us. We’re here to help your trial show accurate, reliable, and valid results.

HSRL Specializes in Histopathology & Specimen Storage Services


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