Through the continuous updating and iteration of technology, HUABIO Cytokeratin antibodies can be used without antigen repair to help researchers ensure IHC verification data while saving precious time.
Why is IHC-P Testing Without Antigen Retrieval Important?
One of the most significant challenges in immunohistochemistry (IHC-P) has been maintaining both good morphology and the immunoreactivity of antigens in tissue sections. Fixation is used in IHC-P to preserve tissue structure and to preserve antigenicity of the target protein. Fixatives crosslink the proteins in the tissue, helping to maintain the three-dimensional structure of the tissue, which allows for better visualization of the target protein. The fixative also helps to preserve the target protein's epitopes, which are the specific sites on the protein that the antibody will bind to during the staining process. Fixation, however, induces major artifacts, masking antigens due to cross-linking among the amino-acid residues of proteins. Antigen retrieval techniques were devised to unmask the hidden antigen epitopes and recover immunoreactivity. When using Heat-induced Epitope Retrieval (HIER), false-positive staining and nonspecific background staining occur as the heating conditions become more severe. It has been suggested that antigen retrieval can also denature proteins in tissues, just like in many other protein inactivation processes.
The benefits include:
- Reduced tissue degradation: Antigen retrieval techniques such as boiling or microwave heating can cause tissue degradation and loss of morphological features, especially in paraffin-embedded tissue samples. Omitting antigen retrieval can help preserve the tissue structure.
- Retention of antigenicity: Some antigens, especially in formalin-fixed, paraffin-embedded tissue samples, may already be optimally exposed and accessible for antibody binding, so antigen retrieval may not be necessary. Omitting antigen retrieval can help to avoid altering the native conformation of the antigen and preserve its antigenicity.
- Reduced non-specific binding: Antigen retrieval techniques can sometimes cause non-specific binding of the primary antibody to tissue components. This can lead to false positive staining results. Omitting antigen retrieval can help to reduce the risk of non-specific binding.
- Simplified staining procedure: Antigen retrieval procedures can be time-consuming and technically demanding, adding steps and complexity to the IHC staining procedure. Omitting antigen retrieval can simplify the staining procedure, reducing the risk of technical errors and making it easier to perform.
- Cost-effectiveness: Antigen retrieval reagents and equipment can be expensive, and the use of antigen retrieval may also require additional time and resources, such as prolonged incubation times or additional washing steps. Omitting antigen retrieval can help to reduce the cost and resource requirements of IHC staining.
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Sample Protocol
Formalin Fixation
Tissue was fixed by 10% Neutral Buffered Formalin (NPF) within 30 min after collection.
Tissue was fixed for 24-48h, then dehydrated by dehydrator, and then embedded into wax blocks.
Sections
Tissue was cut in 3μm sections.
Hydration
The slides were dewaxed with xylene and washed with xylene | 20 min- xylene || 20 min - anhydrous ethanol | 3 min - anhydrous ethanol || 3 min - 95% alcohol 3 min - taper for 3 minute. The slides must be deparaffinized and rehydrated.
Place the slides in a rack. Perform the following washes using a Coplins jar:
Xylene, 3x3 min; 100% ethanol, 3 min; 95% ethanol, 3 min; 95% ethanol, 3 min; 50% ethanol, 3 min;
Place slides in running cold water to rinse off ethanol.
Keep the slides in the tap water until ready to perform antigen retrieval. Do not allow slides to dry out.
Endogenous Peroxidase Blocking
The slides were blocked in a 3% hydrogen peroxide solution, incubated in the dark at room temperature (RT) for 10 min and rinsed with running water for 3 min.
Antigen Retrieval
Tris-EDTA (PH9.0) or Citric Acid (PH6.0) at 90-95℃ for 20 min.
Without Antigen Retrieval
Rinse slides in Phosphate Buffered Saline (PBS) for 20 min and at RT.
Blocking and Staining
Block in 10% normal goat serum with .1% Triton X-100 in Tris Buffered Saline (TBS) for 20 min at RT.
Apply primary antibody diluted in TBS with 1% BSA for 1h at RT.
Rinse 3x3 min TBS 0.1% Tween20 with gentle agitation at RT.
Apply secondary antibody (HA1119) for 30 min at RT.
Rinse 3x3 min TBS 0.1% Tween20 with gentle agitation RT.
Develop with chromogen within 3 min at RT.
Counterstain, dehydrate, clear and mount in slide stainer workstation
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