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Antigen Masking During Fixation and Embedding

Formalin fixation and embedding in paraffin is still the best and most widely accepted method for preserving tissue samples. The reason is simple: this is a method that best preserves the tissue and cells (especially nuclear) morphology. However, in most of cases, one would like to perform immunostaining on collected and preserved tissues. While proper antigen retrieval can be achieved with 2100 Retriever and R-Universal buffer, it is important to know what stages of sample preparation are critical for antigen preservation.

A standard process of preparing a tissue sample for antigen retrieval and immunostaining involves a sequence of stages: formalin fixation/dehydration in ethanol and xylol/embedding in hot paraffin (54-60oC). When sections are being cut, the same stages (except the fixation) are performed in reverse order to allow sections to spread, deparaffinise and re-hydrate.

It is known that, for a majority of antibodies, these operations mask the epitope due to molecules cross-linking by fixation and their aggregation/wrong folding due to the heat. A recent study of Scalia et al (2017) on how important each operation is for antigenicity of the molecules allows to make some critical conclusions about what really matters when fixing and embedding tissues.

The researchers have used cryostat sections and subjected them to all the operations of tissue processing at embedding. A panel of antibodies to various antigens (most of them -critical for diagnostics) were tested for antigen recognition after each section treatment.

Examples of antigen masking after formalin fixation for 30 min and 24 hours, as well as during subsequent dehydration and embedding procedures. Part of the image from Scalla et. al. (2017). To see the full figure at the source, please follow the link

The conclusions are worth reviewing and incorporating into protocols:

- Length of fixation in formalin (paraformaldehyde): practically any treatment with formalin suppressed immunoreactivity of practically all epitopes (times of 30 min – 72 hours at room temperature were tested). However, after antigen retrieval, the epitopes were successfully recovered but the longer fixation times in general benefited the quality and intensity of post-retrieval staining. It appears that, quite unexpectedly, 48 hours of fixation is better than 24 hours, and definitely better than short fixation. For some antigens it was a less pronounced effect, for others – more, but was observed for all tested antibodies. The tested fast fixation in hot (+60C) formalin, from 30 min to 2 hours resulted in lower post-retrieval signal for most antigens.

-IF is more sensitive to the quality of tissue processing. When signals obtained with IF and normal IHC were compared the effects of fixation were more pronounced for IF. This is in agreement with the common sense, as IF detection is more quantitative, and the signal is proportional to amount of available antigen, while IHC has a certain plateau level, when a 2-5x difference in antigen results in more or less the same signal.

- Alcohol that is used in the dehydration steps may increase immunoreactivity suppressed by formalin fixation. It is likely due to the somewhat denaturating effect of alcohol on proteins. From this point, step-wise rehydration of sections in sequential lowering alcohol steps may prepare the section to better antigen retrieval (this was not shown directly). We saw that some denaturation agents, such as SDS or Tween-20, do increase the signal similar to alcohol.

- Xylene and Hot Paraffin. For some antigens to a greater extent, for others – lesser, but xylene reduced the reactivity of antigens, which remained after the formalin fixation. However, the most critical step for antigen “masking” is embedded in hot paraffin in tissue sections in a hot oven.

Examples of antigen masking after formalin fixation for 30 min and 24 hours, as well as during subsequent dehydration and embedding procedures. Part of the image from Scalla et. al. (2017). To see the full figure at the source, please, follow the link.

Please, be advised that most of issues with antigenicity of molecules in FF/PE sections are successfully resolved by processing the tissue sections in R-Universal buffer. This is especially important when a multicolour immunofluorescent staining is planned, as antigens that normally would recover different buffers (High, Low etc.) could all be recovered in the same one time treatment.

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