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Maintenance of Fluorescence During Paraffin Embedding of Fluorescent Protein-Labeled Specimens

Ouyang Zhanmu1,2, Peilin Zhao1,2, Yang Yang1,2

  • 1Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, China.

Frontiers in Neuroscience
|August 10, 2019
PubMed

Insights

Researchers optimized paraffin embedding for fluorescent microscopy. Using tertiary butanol (TBA) instead of ethyl alcohol during dehydration significantly preserves green fluorescent protein (GFP) fluorescence in biological specimens.

Area of Science:

  • Neuroscience
  • Microscopy
  • Biotechnology

Background:

  • Paraffin embedding is a standard technique for preparing biological specimens for microscopic imaging.
  • Traditional paraffin embedding causes significant fluorescence quenching, limiting its use with fluorescent labeling methods like green fluorescent protein (GFP).

Purpose of the Study:

  • To investigate the mechanism of fluorescence quenching during paraffin embedding.
  • To optimize the paraffin embedding process for improved preservation of fluorescence intensity.
  • To develop a method compatible with fluorescent labeling techniques.

Main Methods:

  • Investigated the cause of fluorescence quenching during paraffin embedding, identifying dehydration as the primary factor.
  • Modified the dehydration step by replacing ethyl alcohol with tertiary butanol (TBA).
  • Evaluated fluorescent and morphological preservation in mouse and rat brain samples.

Main Results:

  • The modified TBA dehydration method increased fluorescence intensity 12.08-fold compared to the traditional method.
  • Uniform fluorescence was maintained throughout whole mouse brains, clearly visualizing dendrites, spines, and axon terminals.
  • Successfully embedded and visualized fluorescently labeled neuronal tracts (AAV and tdTomato) in whole rat brains.

Conclusions:

  • Dehydration using ethyl alcohol causes significant green fluorescent protein (GFP) denaturation and fluorescence quenching.
  • The modified paraffin embedding process using tertiary butanol (TBA) effectively preserves fluorescence intensity and morphology.
  • This optimized method is compatible with various fluorescent labeling techniques and offers a significant advancement for neuroanatomical studies.
Keywords:
dehydrationgreen fluorescent proteinparaffin embeddingpreservation of fluorescencewhole brain

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