A novel method for reliable nuclear antibody detection in tissue with high levels of pathology-induced autofluorescence

Immunofluorescence is the basis for many techniques used to quantify phenomena in neuroscience research, in both normal and pathological tissue. Autofluorescence (non-specific, broad spectrum background fluorescence) is an unfortunate consequence of damage to brain tissue. Damage-induced autofluores...

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Bibliographic Details
Published in:Journal of neuroscience methods 2009-12, Vol.185 (1), p.45-49
Main Authors: Spanswick, Simon C., Bray, Doug, Zelinski, Erin L., Sutherland, Robert J.
Format: Article
Language:English
Subjects:
Adrenalectomy
Animals
Antibodies - analysis
Antibodies - metabolism
Benzimidazoles - analysis
Benzimidazoles - metabolism
Biomarkers - analysis
Biomarkers - metabolism
Brain Infarction - diagnosis
Brain Infarction - metabolism
Brain Infarction - physiopathology
Cell Nucleus - metabolism
Cell Nucleus - pathology
Dentate Gyrus - metabolism
Dentate Gyrus - pathology
Disease Models, Animal
False Positive Reactions
Fluorescence
Fluorescent Dyes - analysis
Fluorescent Dyes - metabolism
Image Processing, Computer-Assisted - methods
Immunohistochemistry
Immunohistochemistry - methods
Indoles - analysis
Indoles - metabolism
Ki-67 Antigen - analysis
Ki-67 Antigen - metabolism
Male
Microscopy, Confocal - methods
Neurogenesis
Neurons - metabolism
Neurons - pathology
Predictive Value of Tests
Rats
Rats, Long-Evans
Sensitivity and Specificity
Stroke
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Description
Summary:Immunofluorescence is the basis for many techniques used to quantify phenomena in neuroscience research, in both normal and pathological tissue. Autofluorescence (non-specific, broad spectrum background fluorescence) is an unfortunate consequence of damage to brain tissue. Damage-induced autofluorescence potentially confounds analyses of tissue labeled with fluorescent markers in many experiments. This is especially problematic in protocols that utilize co-localization methods such as BrdU/NeuN in which autofluorescence might lead to overestimates of the number of double-labeled cells. Techniques to reduce autofluorescence are variable and relatively ineffective in damaged brain tissue. Here we show using confocal microscopy that damage-induced autofluorescence does not co-localize with the nuclear specific markers DAPI or Hoechst. Further co-localization of nuclear markers such as Ki67 or BrdU/NeuN with DAPI or Hoechst should serve to help discriminate between intended and spurious fluorescent signal.
ISSN:0165-0270
1872-678X
DOI:10.1016/j.jneumeth.2009.09.007