- Proteins and Peptides
- Lysates and Cell Lines
Sample Preparation for IHC Experiments:
Tissue is prepared and preserved through paraffin embedding or cryopreservation (freezing). Often the preservation method is closely associated with the type of fixation. Formalin-fixed tissues are commonly paraffin-embedding following fixation, while frozen tissue sections are fixed with alcohol following cryopreservation. Each method has distinct advantages and disadvantages. While paraffin embedding is thought to better preserve morphological details, cryopreservation is considered to better preserve enzyme and antigen expression. The optimal method for each experiment should be determined by considering the nature of the antigen, its subcellular location, and desired method of detection, among other factors.
Should I fix the tissue before paraffin embedding?
How long should I fix the tissue before embedding?
Should I dehydrate the tissue before paraffin embedding?
What temperature should I heat the paraffin to?
Can I store paraffin-embedded tissue at room temperature?
How should I freeze my tissue samples? Frozen tissue is prepared by tissue immersion in isopentane or liquid nitrogen. Snap freezing is also used to freeze tissue.
When do I fix my samples if during cryopreservation? After cryopreservation and tissue sectioning, slides are removed from the freezer and allowed to warm at room temperature. The slides are then immersed in fixative (usually alcohol or acetone).
Should I run an antigen retrieval step on frozen tissues? Fixation in alcohol avoids the need for epitope retrieval required when tissue is fixed using formaldehyde. Alcohol fixation does not create crosslinks, therefore antigen retrieval is not required.
How should I store frozen tissues? Frozen tissue sections can be stored at -80 °C for up to 1 year.
Should I use frozen or paraffin-embedded tissue to study phosphorylation? Highly sensitive proteins which are susceptible to rapid deterioration, such as post translational modifications, can be better preserved by snap freezing. Formalin fixation can alter the expression of post-translationally modified proteins.
Is paraffin-embedded tissue better than frozen tissue? This is dependent on the details of your experiment. Formalin-fixed paraffin-embedded tissue can be stored long term at room temperature whereas frozen tissue can only be stored for up to one year at -80 °C. Frozen tissues can be affected by the formation of ice crystals, which can affect subcellular detail and impact IHC staining. Another potential drawback to frozen tissue is the thickness of the section. Compared to paraffin-embedded sections, frozen tissues are thicker, lowering microscopic resolution and the ability to capture tissue morphology in detail. However, cryopreservation is thought to better preserve antigen and antigenicity. The study of post-translationally modified protein, DNA, or RNA is also recommended on frozen tissue.