AAT Bioquest

What are the factors that will affect the rate and effectiveness of tissue fixation?

Posted December 8, 2022


Factors that affect the rate and effectiveness of tissue fixation include: 

Tissue thickness – The correct thickness of the tissue is critical for efficient fixation. A large sample size prevents the fixative from penetrating through the tissue and reaching the deeper areas. This results in autolysis of the epithelium. Sectioning the tissues thinly at about 4 mm to 6 mm allows the fixation to penetrate quickly and completely. 

pHpH is another important factor that affects the rate and effectiveness of tissue fixation. Excessive acidity promotes the formation of formalin-heme pigment deposits in tissue and may lead to structural distortions and protein denaturation. Cell fixation is most efficient when carried out around neutral pH between 6 and 8.

Temperature – Temperature of the fixative also affects the rate and effectiveness of tissue fixation. Increasing the temperature increases the rate of fixation but it also increases the rate of autolysis, with thermal denaturation occurring at extreme temperatures. Lowering the temperature decreases the rate of diffusion, which increases the penetration time. 60°C is the ideal temperature with formaldehyde fixatives.

Volume of fixative – A 10:1 ratio of fixative of tissue is ideal for efficient tissue fixation. Higher or lower ratios will impact both the rate and effectiveness negatively. 

Concentration of fixative – Rate and effectiveness of tissue fixation will also be affected if the concentration of the fixative is too high or too low. Higher concentrations of the fixative can damage cellular structures and hinder enzyme activities. If the concentration is too low, the fixative is likely to be exhausted before the process is complete. The ideal concentration is different for different fixatives. 

Length of Fixation – Longer fixation time results in samples becoming brittle because of excessive cross-linking. Shorter fixation time may not allow for full penetration in tissues and cross-linking. The optimum fixation time is different for different types of tissues and is experimentally determined for each tissue type.

Additional resources

Fixation dynamics of beneficial alleles in prokaryotic polyploid chromosomes and plasmids

Buffers and Lab Consumables

ReadiUse™ 4% formaldehyde fixation solution