Tissue clarification is the third step in processing.
Having finished the previous steps (dehydration) the tissue has completely lost its water. but still it can not be replaced by Paraffin because paraffin and ethanol are immiscible. So we need an intermediate environment in which paraffin and ethanol could be mixed separately.
By dehydrating, the tissue loses its transparency, so solutions must Be able to remove the alcohol in the tissue to make it transparent. Meaning that refractive index to be equal to that of protein. These solutions Must have the ability to be dissolved in the alcohol used in the previous step. Such as hydrocarbon solvents such as chloroform, carbon tetrachloride, Xyl or benzene.
Note: Pure xylene is used in most laboratories. as it has better performance,it is fast and cheap enough . and does not make any contamination.
The method is that after absolute alcohol, 2 or 3 containers of xylene are placed.
And the time required for clarification is 4 hours Which can be doubled if necessary. 80/20 Ethanol-isopropanol or Pure isopropanol mixture can be used instead of xylene due to its toxicity characteristics and bad smell. but For this method to be successful, we need paraffin at high temperature to extract isopropanol from the sample.