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Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations
- Source :
- Brain Structure and Function. 223:987-999
- Publication Year :
- 2017
- Publisher :
- Springer Science and Business Media LLC, 2017.
-
Abstract
- The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.
- Subjects :
- Male
0301 basic medicine
Time Factors
Histology
Confocal
Fluorescent Antibody Technique
Immunofluorescence
Article
Mice
03 medical and health sciences
chemistry.chemical_compound
Imaging, Three-Dimensional
Lectins
medicine
Animals
Sodium dodecyl sulfate
Acrylamide
Microscopy, Confocal
Chromatography
Dose-Response Relationship, Drug
Staining and Labeling
medicine.diagnostic_test
biology
General Neuroscience
Brain
Lectin
Fluorescence
Staining
Mice, Inbred C57BL
Parvalbumins
030104 developmental biology
Monomer
chemistry
biology.protein
Anatomy
Subjects
Details
- ISSN :
- 18632661 and 18632653
- Volume :
- 223
- Database :
- OpenAIRE
- Journal :
- Brain Structure and Function
- Accession number :
- edsair.doi.dedup.....383c54af76a054fbe8c13dffa1996b97