Maintenance Of Fluorescent Protein-Labeled Specimens

Paraffin embedding has been a widely-used method for histomorphology. Embedding is more efficient than other methods and has good sectioning properties, but it also reduces fluorescent proteins. It results in a low fluorescent signal, making detection difficult. GFP and its variants, which include the popular enhanced green fluorescent protein (EGFP), and the enhanced yellow fluorescent protein (EYFP), can be used in many areas, such as neuroscience and drug screening, and cancer research. 

Transgene and virus tracking with GFP-based reporter protein provide an easy way to study neuronal morphology, disease progression, and structure and function. To detect GFP proteins, antibodies are used to combine paraffin embedding and fluorescent labeling techniques. This compromising staining technique can lead to false negatives and false positives. It is therefore urgent to develop a paraffin embedding technique on paraffin-embedded tissue compatible with fluorescent protein labeling. 

Paraffin Embedded Tissue Blocks

Image Source: Google

Many researchers believe that paraffin embedding can be halted by high temperatures and chemical fixing reagents. The best way to detect paraffin-embedded GFP in paraffin is to stain them with immunofluorescence (IHC) or immunohistochemistry. It is not clear what caused fluorescence quenching in paraffin embedding. GFP is used to label biological structures and molecules. It has become necessary to combine these with traditional histochemical methods. 

The embedding protocol has been empirically optimized by researchers to enhance fluorescence preservation in paraffin-embedded samples. Modified versions of the ethanol fixation protocol at 4°C were most effective, allowing GFP visualization. However, not all samples can be effectively fixed with ethanol. GFP was detected but no details about the fine structure of the sample were available. Fluorescence quenching is still an issue, so it's difficult to think about the use of GFP for paraffin embedding.

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