During tissue preparation, antigens are masked by chemical reagents, and some antigenic peptide chains are distorted due to heat, making them undetectable in immunohistochemical staining. To solve these problems, the process of re‑exposing or correcting these antigens using chemical reagents and heat is called antigen retrieval. Buffers such as citrate, EDTA, or Tris can re‑expose formalin‑masked antigens under heating conditions without damaging antigenic epitopes, thereby improving antigen detection rate, reducing background staining, and enhancing diagnostic accuracy.
Before the advent of heat‑induced antigen retrieval, epitope retrieval induced by enzymes such as trypsin, proteinase K, and pepsin was the most commonly used method. The Trypsin Antigen Retrieval Kit can expose antigenic epitopes in samples such as paraffin sections, greatly improving immunostaining results. In general, paraffin sections require antigen retrieval, while frozen sections may not. Antigen retrieval can enhance immunostaining of paraffin sections and also improve staining of frozen sections to varying degrees. Antigen retrieval can be considered when immunostaining of frozen sections is unsatisfactory. This reagent is for research use only. Not for clinical diagnosis or other applications.
Materials Provided by User
1. Graded ethanol, double‑distilled water or deionized water, immunostaining washing solution
2. Heating equipment
Protocol (For Reference Only)
(I) Paraffin Sections
1. Deparaffinization and rehydration
① Deparaffinize with xylene or deparaffinization clearing solution 3 times, 3-5 min each.
② Dehydrate with absolute ethanol 2 times, 3-5 min each.
③ 95% ethanol, 3-5 min.
④ 90% ethanol, 3-5 min.
⑤ 80% ethanol, 3-5 min.
⑥ 70% ethanol, 3-5 min.
⑦ Rinse with distilled water 2 times, 3-5 min each.
2. Antigen retrieval: Incubate sections in Trypsin Solution at 37℃ for 10-25 min.
3. Wash 1-2 times with TB Washing Buffer and tap water, 3-5 min each, until thoroughly rinsed.
4. Proceed with subsequent immunostaining steps.
(II) Frozen Sections
1. Wash sections with immunostaining washing solution or tap water for 5 min.
2. Antigen retrieval: Incubate sections in Trypsin Solution at 37℃ for 10-25 min.
3. Wash 1-2 times with TB Washing Buffer and tap water, 3-5 min each, until thoroughly rinsed.
4. Proceed with subsequent immunostaining steps.
(III) Other Samples
Follow the procedure for paraffin or frozen sections as appropriate.
Precautions
1. Allow Trypsin Solution to equilibrate to room temperature before use; avoid repeated freeze‑thaw cycles.
2. The optimal incubation time in Trypsin Solution must be determined empirically for different samples and target proteins.
3. For your safety and health, wear a lab coat and disposable gloves during operation.
4. Use the reagent promptly after opening to avoid compromising experimental results.
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