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 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.
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.
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