IHC Fixation of Samples: Tips and Tricks: Novus Biologicals

IHC Tissue Microarrays

Related Links

Sample Preparation

Blocking Non-Specific Binding

Primary Antibody Selection

Protocols

IHC-P troubleshooting guide

IHC-F troubleshooting guide

IHC epitope retrieval (HIER)

IHC epitope retrieval (PIER)

All protocols

Fixing Samples for IHC

Fixation preserves tissue for future analysis by preventing autolysis (cell destruction mediated by a cell’s own enzymes) and decay. For the best results, tissue should undergo rapid and uniform fixation. This can be achieved by two methods: whole animal perfusion or tissue/organ immersion in fixative. The choice of fixative, and duration and method of fixation can impact preservation and tissue integrity, so ideal fixing conditions should be determined to preserve tissue optimally for each experiment.

Fixation Methods

Fixation Method	What is it	When to use it	Advantages	Disadvantages
Whole Animal Perfusion Fixation	Perfusion of 4% paraformaldehyde solution through the vascular system	For large, intact tissue of small animals.	Rapid dispersion of fixative throughout the animal via the vascular network results in uniform fixation of large organs.	Technically challenging Time consuming
Immersion in Fixative	Immersion of tissue at room temperature in a fixative volume 50-100 times greater than the volume of the tissue.	For small pieces of dissected tissues (less than 10 mm).	Quick Technically simple.	May result in incomplete fixation of large tissue due to failure of fixative to penetrate all regions of tissue at the same rate.

Tips About Fixation

When do I fix my samples? It is recommended fixation of tissue occurs immediately following death or dissection. Autolysis and tissue damage occur when blood flow is interrupted post-mortem. Fix tissues immediately to improve tissue preservation.
At what temperature should I fix? Fix tissues at room temperature. Higher temperature increases fixative penetration, but also accelerates the rate of enzyme-mediated tissue degradation (autolysis).
In what volume do I fix? Immersion in a fixative volume 50-100X greater than the volume of the tissue is recommended. For optimal fixation following whole animal fixative perfusion, overnight immersion follow dissection is recommended.
How long do I fix? Overnight fixation for 4-24 hours in recommended. However, optimal fixation conditions should be determined to prevent underfixation or overfixation (read more below).
How should I store formalin fixed tissues? Tissues fixed with formalin should be stored in 70% ethanol if storage is required prior to paraffin embedding.

Overfixation versus Underfixation

The duration of fixation can impact sample integrity. Optimal fixing conditions should be determined for each tissue and antigen to prevent underfixation or overfixation.

	What is it	Effect
Underfixation	Incomplete fixation, immersion in fixative too short	Can result in autolysis and target protein degradation Can produce staining artifacts when dehydrating alcohols are used on under-fixed tissues
Overfixation	Excessive fixation, immersion in fixative too long	Can result in epitope masking Can produce strong non-specific staining.

Choosing a Fixative

The choice of fixative can have a significant impact on the ability to preserve morphology and generate protein expression data.

	Advantage	Disadvantage
Formalin	Better preservation of tissue morphology Long term storage if paraffin embedded.	Can mask epitope and reduce antigenicity. Can affect expression of post translational modifications
Alcohol	Better preservation of antigenicity. Better suited for the study of DNA, RNA, and post translational modifications.	Can distort nuclear and cytoplasmic detail.

Formalin

Formalin is a common fixative widely used in many research labs. Although many fixatives are available, formaldehyde is an appropriate starting point and often sufficient for most IHC applications. However, it is not universal.

How does Formalin work? When introduced to sample, formaldehyde solution diffuses and reacts with amino acids in sample to form reactive groups. These reactive groups combine with one another to form methylene bridges (crosslinks).

Do I have to prepare fresh formaldehyde solutions? It is recommended to prepare formaldehyde solutions fresh or aliquot and freeze them to reduce the possibility of artifacts. Unbuffered formalin and formaldehyde solutions can oxidize slowly, forming formic acid which can react with hemoglobin in tissue to form a black/brown artifact, especially in blood-rich tissues.

Do I need an antigen retrieval step? Formalydehye fixation can mask epitope and limit antibody-epitope binding. To unmask epitope and restore antigenicity an antigen retrieval step, such as heat induced epitope retrieval (HIER) or proteolytic induced epitope retrieval (PIER), prior to IHC staining is recommended.

Can I study phosphorylated proteins in formalin fixed tissues? Induction of intracellular translocation of phosphorylation-dependent epitopes from the membrane to cytoplasm can occur in formalin fixed tissues. If a phosphorylated protein will be investigated, ice cold methanol or ethanol should be used as fixatives. However, formalin fixed tissue can be used in some cases.

Should I use formalin or alcohol to fix? Formalin is thought to preserve tissue morphology better than alcohol fixatives. However, alcohol fixation better preserves antigen and antigenicity.

Alcohols

Ethanol and methanol are the most popular alcohols used for cell and tissue fixation.

How does alcohol work? Ethanol and methanol replace water in the tissue, exposing the internal hydrophobic proteins and breaking hydrophobic bonds to alter tertiary structure. Alcohol is considered a precipitating fixative.

What concentration of alcohol should I use? A concentration greater than 70% for ethanol and 80% for methanol is required to commence fixation.

Do I need an antigen retrieval step? An antigen retrieval step is not recommended on alcohol fixed tissues since it is often too harsh and can impact tissue integrity.

Back to Sample Preparation Forward to Blocking Non-Specific Binding

IHC/ICC Sample Fixation (Formalin vs. Alcohol)