This to educate on what proportion of substances used to prepare solution to treat the animal tissue for histological microscopic examination.
For histology the standard preferred fixative is 10% neutral buffered formalin (NBF).
"Neutral buffered formalin, usually simply shortened to NBF, has become the standard fixative for use in a diagnostic setting. It is more effective than the simple formalin mixtures as the phosphate salts present make it unlikely that erythrocytes will be damaged, and the neutral pH inhibits the formation of formalin pigment".
As indicated above, for routine histology the standard fixative is a 10% solution of neutral buffered formalin (NBF).
If you do not purchase this commercially, then you should be aware that the concentrated formaldehyde stock used to make NBF actually contains 37% formaldehyde w/v. This means that the standard 10% NBF used to fix tissues actually contains 3.7% formaldehyde w/v. This is an important point if you are mixing up the 10% NBF from a recipe such as this:
- 4.0 gm Sodium phosphate, monobasic
- 6.5 gm Sodium phosphate, dibasic
- 100.0 ml Formaldehyde, 37%
- 900.0 ml Distilled water
- Adjust to neutral pH.
For optimum preservation, tissues should be less than 1 cm in greatest thickness. The ratio of tissue to NBF should be 1:10, though it is not uncommon to receive adequately fixed biopsy specimens at a 1:5. Most tissues prepared this way will be fixed within 24 hours. Thinner, non-bloody tissues could be adequately fixed as early as 4-6 hours, but bloody tissues like thick sections of congested spleen take considerably longer to fix. For whole brains, particularly those of large animals, fixation time can be substantially prolonged, and I prefer to use a home-made 50% NBF to accelerate the process. In general, eyes fare best if they are intact and fixed in NBF, though some specialized analyses fare better with Bouin's or Zenker's solution. Many tox path folks prefer to fix testes in Bouin's as well.
As above, I use neutral buffered formalin, but make sure to change to 70% ethanol after about 18-24 hours fixation to allow optimal IHC staining.
As indicated above, for Standard Histological Examination, we use the NBF neutral buffered formalin with 10% solution.
10% NBF is the common fixative used for routine uses.
But for IHC 4% paraformaldehyde has an advantage.
Bouins fixative is a rapid fixative preferred to maintain the integrity of the tissue architecture.
For EM studies, gluteraldehyde based fixatives are used.
4% paraformaldehyde is the best - if you want samples to be useful for immunostaining it is important not to leave it in paraformaldehyde for too long (max 24hrs) and process and embed in paraffin blocks asap.
For histology staining also 4% paraformaldehyde and samples can be stored like this for a good while before processing and embedding.
10 percent Neutral buffered formalin is the preferred. fixative.used. for standard
Histological
10% neutral buffered formalin for 48 hours, possibly diluted in NaCl solution to create a similar osmotic environment between tissues and fixative.
For histological purpose 10% NBF is best and for IHC purpose 4% PFA is better option.
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