IHC Sample Preparation (Frozen vs. Paraffin)

IHC Tissue Microarrays

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Sample Fixation

Blocking Non-Specific Binding

Primary Antibody Selection

Epitope Retrieval

IHC Detection


IHC-P Protocol

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IHC Epitope Retrieval (HIER)

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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.


Paraffin-embedded Tissue

Frozen Tissue

Fixation Prior to embedding After sectioning
Sectioning Microtome Cryostat
Storage Long-term, multiple years at room temperature Short-term, 1 year at -80 °C
Advantages Preserves tissue morphology Preserves enzyme & antigen function
Limitations Overfixation can mask the epitope Ice crystal formation may negatively affect tissue structure


Paraffin-embedded Tissue

Should I fix the tissue before paraffin embedding?

The best option to preserve tissue for extended time is paraffin embedding. Immediate perfusion or immersion of tissue in fixative prior to embedding is necessary to prevent tissue destruction and ensure preservation.

How long should I fix the tissue before embedding?

To achieve the best results, tissue is fixed for 4 to 24 hours. Tissue fixation for more than 24 hours may result in overfixation, masking antigen and limiting antibody-epitope binding in IHC staining.

Should I dehydrate the tissue before paraffin embedding?

The immiscibility of paraffin and water necessitate the need for tissue dehydration prior to the addition of molten paraffin wax. Submersion of tissue in increasing concentrations of alcohol results in alcohol penetration of tissue and replacement of water with alcohol. Because alcohol shrinks and hardens tissue through dehydration, the length of immersion must be closely monitored. After dehydration, immersion in xylene removes any excess or remaining alcohol and the tissue is ready for embedding.

What temperature should I heat the paraffin to?

Typically, paraffin is heated to 60 °C for embedding, added to tissue, then allowed to harden overnight.

Can I store paraffin-embedded tissue at room temperature?

Tissues embedded in paraffin blocks or paraffin embedded tissue sections on slides can be stored at room temperature.


Frozen Tissue

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.

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