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The 4 steps to avoiding major histology pitfalls

Monday, June 2nd, 2014

 

Avoid major, irretrievable mistakes in animal histology by taking the following steps!

1. Always label cassettes with a #2 pencil

Use a pencil or a marker made for histology on cassettes. Sharpie markers are not solvent resistant, and labels written in Sharpie are removed during routine processing, meaning that by the end of processing you have a bucket full of cassettes with no IDs. Even pens marketed for histology purposes do not always resist solvents. If you have any doubt about a marker you plan to use, we would be happy to run an empty, labeled cassette through the processor as a trial before you submit tissues. The safest plan is to use a sharp #2 pencil.

2. Save all tissues

Once you discard tissues at necropsy, they are gone forever! Even if you don't plan to use the tissues, collect all organs into formalin. You can always discard the extra tissues later at the end of your study when your paper is published and you know that you are sure you don't have a use for the tissue anymore. Definitely save any organs or tissues that look different than normal and that you plan to ask us about.

3. Use more formalin!

Use a 15:1 volume of formalin to tissue. Then add a little more formalin! One common problem that we see in research animal histology is tissue that has not been fixed adequately. Tissue that is preserved in too low a volume of formalin does not fix before it autolyzes, and tissue morphology can be destroyed irretrievably. Experiments can be ruined because the tissue was put in too small of a volume of formalin. Avoid conical tubes, because the tissue sits at the bottom of the tube, filling up the conical bottom, and gets little exposure to the formalin above. Use flat bottomed centrifuge tubes or use histology containers. Large masses or organs from any species larger than a mouse should be cut before fixation to no more than 0.5 cm width. Formalin must penetrate the tissues before it fixes them. Tissue that is incompletely fixed by formalin and then put on the tissue processor will complete its fixation by coagulative fixation from the alcohols during processing. Morphology and IHC reactivity can be affected by incomplete fixation and by fixation by two different methods. While there is no issue using PFA to fix tissues, it must be made up fresh every time, and large volumes are more difficult to make. We recommend formalin over PFA for ease of use and for obtaining suitable volumes to completely fix tissues.

 

4. Did you collect lymph node? Are you sure?

The most commonly mistaken organs are lymph nodes. Researchers often think they have collected lymph nodes to find later on histology that they inadvertently collected salivary glands or fat lobules. Double check that you collected the organs you intended to collect.  We offer training in anatomy and tissue collection. Also refer to our list of recommended reading for anatomy references. No matter the organ you are collecting, we recommend a practice collection the first time to be sure you are confident in identifying, collecting, and preparing the tissue for histology.