DEPARAFFINIZATION PRIOR TO IMMUNOHISTOCHEMISTRY STAINING
The process explained here is protocol for deparaffinization prior to immunohistochemistry staining of paraffin-embedded tissue.
Paraffin-embedded tissue is a tissue sample that is preserved using paraffin. Samples can also be preserved by frozen methods such as the use of liquid nitrogen or by lyophilization technique. However, most biological samples including spleen, intestine and lymph nodes to mention a few can be preserved and prepared prior to staining and microscopy using paraffin. Prior to carrying out immunohistochemistry, the paraffin-embedded tissue must be deparaffinized in order to proceed with the immunohistochemistry analysis. Other processes of the immunohistochemistry technique include antigen retrieval, permeabilization and blocking of non-specific binding, antibody staining, antigen detection and mounting of the stained slide using specific gel. Microscopical analysis using fluorescence microscope or confocal microscope is the final stage of the immunohistochemistry analysis; and microscopy allows the scientist or researcher to visualize the internal structures and localization of the antigen or molecule of interest within the stained tissue sample. Generally, deparaffinization removes the wax that covers the embedded tissue sample. The processes involved in the immunohistochemistry staining of paraffin-embedded tissue are highlighted in this section.
Deparaffinization and rehydration of the paraffin-embedded tissue
Deparaffinization is simply defined as the process of removing the paraffin from the tissue. Paraffin covers the tissue structures and may affect immunohistochemistry analysis, especially by masking or blocking the antigen of interest. Therefore, paraffin must be removed first before proceeding with the experiment. Deparaffinization is achieved by using a series of solvents including xylene, ethanol and water. The paraffin-embedded tissue sample is passed through these solvents at various time intervals; and these steps are explained as follows:
- Mark the sectioned tissue on slide with PAP / pen, and allow to dry. A hydrophobic barrier pen or rubber cement can also be used to mark the sectioned tissue on the slide. This must be done prior to deparaffinization.
- De-wax embedded tissue samples in xylene and rehydrate in graded ethanol as follows:
- Immerse slides in Xylene well and allow for 5 min. Repeat this step for three (3) different times. Always ensure to use fresh xylene at every stage.
- Immerse slides in 100 % ethanol for 5 min. Repeat this step for two (2) different times. Ensure to use fresh ethanol at every step.
- Immerse slides in 90 % ethanol for 5 min. Do this step only once.
- Immerse slides in 70 % ethanol for 5 min. Do this step only once.
- Rinse slides with flowing water for 30 s
- Slides are now ready for next step, which is antigen retrieval. See next section for antigen retrieval steps.