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Fixation, Processing, and Immunofluorescent Labeling of Whole Mount Planarians
David J Forsthoefel1, Kelly G Ross2, Phillip A Newmark3
1Functional and Chemical Genomics Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, USA. david-forsthoefel@omrf.org.
Methods in Molecular Biology (Clifton, N.J.)
|June 20, 2018
Summary
This study details immunofluorescent labeling methods for whole mount planarians, aiding in the study of regeneration mechanisms in Schmidtea mediterranea. Optimized protocols ensure effective antigen detection for novel antibodies.
Area of Science:
- Developmental Biology
- Cell Biology
- Regenerative Medicine
Background:
- Planarian regeneration is a key model for studying tissue repair.
- Immunocytochemistry is crucial for understanding cellular processes in planarians.
- Existing methods require optimization for whole mount labeling.
Purpose of the Study:
- To establish robust immunofluorescent labeling protocols for whole mount planarian specimens.
- To facilitate the study of molecular mechanisms underlying planarian regeneration.
- To provide a framework for optimizing sample preparation for novel antibodies.
Main Methods:
- Whole mount fixation and processing of planarians.
- Application of specific reagents for mucus removal, pigment bleaching, and permeabilization.
- Antigen retrieval techniques and optimization of antibody labeling parameters.
Main Results:
- Detailed protocols for immunofluorescent labeling of whole mount planarians are presented.
- Key steps include fixation, mucus removal, bleaching, permeabilization, and antigen retrieval.
- Recommendations for optimizing protocols for novel antibodies are provided.
Conclusions:
- The described methods enhance the ability to study planarian regeneration at the cellular level.
- Optimized immunofluorescence is critical for identifying specific cell types and molecular markers.
- These protocols will advance research into the fundamental mechanisms of regeneration.
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