Gentle effect of trypsin in antigen retrieval for animal tissue sections

The "Hidden" Antigens in Paraffin Sections

In pathological analysis of animal experiments, tissues are often prepared as paraffin sections and then subjected to immunohistochemical staining to observe the location and abundance of a certain protein in the tissue. However, during the preparation process, formaldehyde fixation causes protein cross-linking, which acts like a "veil" covering the target protein, making it difficult for antibodies to bind. In this case, trypsin can be used for antigen retrieval to moderately open up the cross-linked proteins, allowing antibodies to recognize the target again.

The Principle of Trypsin Retrieval

Trypsin can hydrolyze certain peptide bonds in proteins. For some antigens masked by fixatives, brief trypsin treatment can expose antibody binding sites. This process is somewhat like gently erasing a thin layer covering a painting with an eraser, rather than damaging the entire painting. Therefore, concentration and time are very critical. A trypsin solution at a concentration of 0.05%–0.1% is generally used, acting at 37°C for 10–20 minutes, with specific conditions determined by preliminary experiments.

Which Animal Tissue Samples Are More Suitable

Not all antigens are suitable for trypsin retrieval. Some tissues rich in fibrous components and extracellular matrix, or certain nuclear antigens, may show stronger signals after retrieval. For common animal tissues such as liver, kidney, and intestine, literature reports indicate that trypsin retrieval can yield clearer staining. However, different antibodies, fixation times, and animal species can all affect the results, so generalizations cannot be made.

Precautions During Operation

Trypsin can only be used after sections have been deparaffinized and rehydrated; otherwise, the enzyme cannot penetrate the tissue.

After retrieval is complete, the sections must be thoroughly washed with buffer to remove residual enzyme solution and avoid affecting subsequent antibody reactions.

The temperature should be maintained at around 37°C; excessively high temperatures may damage tissue morphology.

Keep the tissue moist during the process to avoid drying out.

Differences from Heat-Induced Retrieval

Many laboratories now also use citric acid buffer heating for antigen retrieval. In comparison, trypsin retrieval is more suitable for certain antigens that are heat-sensitive or require enzymatic digestion for exposure. The two are not mutually replaceable but are chosen based on the target protein and antibody datasheet. Ordinary experimenters can first consult the antibody datasheet, and if enzyme retrieval is recommended, then choose trypsin.

The Practical Effects of Trypsin Retrieval

In some animal tissue sections, after mild trypsin treatment, originally weak positive signals may become clearer, and background staining may sometimes be reduced. However, excessive retrieval can damage tissue structure and the antigen itself, leading to false negatives. Therefore, before starting formal experiments, conducting small-sample preliminary experiments with different concentrations and times is a reliable method to determine suitability.

Summary

Trypsin not only helps experimenters isolate cells from animal tissues but can also help unveil the "veil" of antigens in tissue sections. As long as concentration, time, and washing steps are well controlled, it can become a gentle and practical tool in animal pathology experiments.