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 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.
Spanswick
Simon C
SC
The Canadian Centre for Behavioural Neuroscience, Department of Neuroscience, University of Lethbridge, Lethbridge, Canada. simon.spanswick@uleth.ca
Bray
Doug
D
Zelinski
Erin L
EL
Sutherland
Robert J
RJ
D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000378:metabolism; D016477:Artifacts; D001562:Benzimidazoles / Q000032:analysis / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D020520:Brain Infarction / Q000175:diagnosis / Q000378:metabolism* / Q000503:physiopathology; D002467:Cell Nucleus / Q000378:metabolism* / Q000473:pathology; D018891:Dentate Gyrus / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D005189:False Positive Reactions; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles / Q000032:analysis / Q000378:metabolism; D019394:Ki-67 Antigen / Q000032:analysis / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D011237:Predictive Value of Tests; D051381:Rats; D020318:Rats, Long-Evans; D012680:Sensitivity and Specificity
D000906:Antibodies; D001562:Benzimidazoles; D015415:Biomarkers; D005456:Fluorescent Dyes; D007211:Indoles; D019394:Ki-67 Antigen; C007293:DAPI; C017807:bisbenzimide ethoxide trihydrochloride