Tips on Fixing Tissue for Histology

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Tuesday 06/01/2010

There has been a great deal of discussion recently concerning the optimal tissue fixation conditions for histology. Fixation is the single most important preparative histological technique. Poor fixation cannot be remedied at any later stage and is the source of most of the problems we see in our lab. In our experience, under-fixation is a much greater problem than over-fixation.

Fixative: 10% neutral buffered Formalin (10% NBF)

  • Formaldehyde based fixatives are superior for morphology, special stains and immunohistochemistry.
  • Paraformaldehyde is unstable and needs to be prepared fresh DAILY. There is no data that paraformaldehyde is superior to formalin.
  • 10% formalin and 4% paraformaldehyde have the same amount of formaldehyde.
  • After fixation, tissue can be stored for 1 to 3 days in 70% ethanol. Please consult resource staff if you need to store fixed tissue for a longer time.

Time: 3-7 days in Formalin

  • Formaldehyde is one of the fastest fixing agents to penetrate but also one of the slowest to fix. It takes days to form stable cross-links.
  • If the tissue is under-fixed, exposure to alcohols can dramatically alter antigen structure, modify morphology, and result in the loss of up to 40% of the protein content from the tissue. These effects are permanent and irretrievable.
  • With antigen retrieval techniques, most antigens are detectable in tissue even after 1 year in formalin.

Volume: Ratio of 20:1 fixative to tissue

  • Fixatives are poor buffers and can become acidic if there is not enough volume.

Size: 2 – 5 mm thick and no larger than 2 cm

  • Tissue thickness and size will affect the rate of fixative penetration.
  • A nickel is 2 mm thick and a postage stamp is about 2 cm square. These are good guides to use when grossing tissue.
  • Cutting encapsulated organs or perfusing lungs can increase the rate of fixation and improve morphology.

Temperature: Room Temperature

  • Lowering the temperature to 4 C significantly decreases the rate of fixation.

Special Note: If you decalcify your bone samples, please speak to Experimental Histopathology about new products that are now available that allow you to decal while you fix. The important thing to remember is that many decalcification reagents contain mild acid that can damage tissue and antigens over time. Samples should be treated only long enough to decal the tissue and then the samples should be thoroughly rinsed and placed back into formalin.

What if your samples need to do double duty?

  1. Pick your most critical technique and fix optimally for that. Then adjust your other applications to fit that fixation.
  2. If one fixation is not compatible with all applications then you must divide tissue accordingly.

Experimental Histopathology suggested the following guidelines:

Formalin fixed for 3 to 7 days:

  • General Histology H&E
  • Most special stains
  • Most IHC
  • DNA (will be fragmented and lower yield)
  • RNA (will be fragmented and lower yield)

Formalin fixation for 24 hours:

  • Micro RNA

OCT-Embedded Frozen:

  • Some special stains (such as Oil Red O)
  • Some IHC (such as CD4 in mouse tissue)
  • DNA
  • RNA
  • Some protein

Snap Frozen:

  • DNA
  • RNA
  • Total protein

Please contact Experimental Histopathology (206-667-6166) if you have any questions regarding these recommendations. In addition, please consult the laboratory before using any formalin, alcohol, or xylene substitutes. Some of these products have variable effectiveness and can damage tissue.

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Rajesh K. Uthamanthil, DVM, PhD, DACLAM

Associate Vice President, Shared Resources