Fixation – Using Paraformaldehyde

Fixation is also to be carefully selected for the techniques in use.  Routine fixation in neuroscience with buffered 4% paraformaldehyde is typical but there are a variety of fixatives and fixation methods. Fresh tissue can be sectioned and post fixed but for good retention of labile protein molecules (such as neurotransmitters), transcardial perfusion with a paraformaldehyde-based fixatives is preferred. Post fixation is also a step, which needs careful control and standardization especially when immunohistochemical labeling is used. Cross-linking fixatives can render antigens unavailable for labeling.
Immersion fixation can cause delays in fixing labile protein in situ and can be seen as blurred nuclear staining and poor chromatin preservation.
Paraformaldehyde is toxic and can cause sensitisation reactions. All preparation of powders should be carried out in a fume hood wearing the appropriate PPE. The powder dissolves most effectively in water heated to 50C and sodium hydroxide pellets added gradually to adjust the pH to 7.4.

Important Notes on Paraformaldehyde:

  • Tissues should not be stored in PFA long term – remove your sample from PFA as soon as possible.
  • Over fixation by leaving tissue/cells in paraformaldehyde for too long renders some antigens irretrievable and increases autofluorescence.
  • Fixation time can vary depending on the antigen of interest and tissue type. It is very important to check you are using the right fixation protocol for your tissue and technique

Other fixatives are used in histology and should be selected to optimise staining and morphological preservation.  Antigen retrieval of paraformaldehyde paraffin embedded tissue is necessary and this can be achieved using enzyme digestion or heat retrieval (for standard solutions see page 25). All times for retrieval and antibody concentration titration must be carried out on a known positive slide to optimise staining prior to application to the test sample.

Information about perfusion fixation vs drop fixing
There are differences in the characteristics of tissues which have been perfused and those which are immersion or drop-fixed. If immunohistochemical labeling using the DAB chromagen, then be aware that drop fixed material will have erythrocytes present which will stain if not adequately blocked with hydrogen peroxide prior to staining. Also nuclear detail can be affected in immersion fixed tissues due to the diffusion of nuclear proteins out of the nucleus as the fixative diffuses into the tissue. Where possible perfusion fixation is highly recommended as this is instantaneous at labile proteins will be fixed in situ.