Triple fluorescence labelling of neuronal, glial and vascular markers revealing pathological alterations in various animal models.
The simultaneous detection of glia, vessels and neurons facilitates insights into the complex chemoarchitecture of the central nervous system. Here, we present a simple, robust and versatile approach for the carbocyanine triple fluorescence labelling of neuronal, vascular and glial markers. The usefulness of this procedure is shown for rat brain tissue under physiological conditions, after traumatic brain injury caused by controlled cortical impact injury, and after stroke following middle cerebral artery occlusion. Moreover, the versatility of the method is verified by its application to sections from old triple transgenic mice with age-dependent beta-amyloidosis and tau hyperphosphorylation in the hippocampus, modelling neuropathological alterations in Alzheimer's disease. To exemplify the usefulness of the approach for analysis of the enteric nervous system, it was applied to whole mounts from the horse intestine. The biotinylated lectin from potato (Solanum tuberosum) is presented as an excellent tool to detect both vessels and microglia. Furthermore, this lectin revealed macrophages after experimental insults, and senile plaques in aged triple transgenic mice. A large portion of astroglia was demonstrated by immunolabelling of glial fibrillary acidic protein. Neurons were detected by monoclonal antibodies directed against neuronal nuclei and, in horse tissues, mouse-anti-HuC/D recognizing a conserved nuclear protein. Confocal laser-scanning microscopy elucidated spatial relationships of the relevant markers and their pathological alterations after experimental insults and in transgenic mice with Alzheimer-like lesions.
Härtig
Wolfgang
W
Paul Flechsig Institute for Brain Research, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany. hartig@medizin.uni-leipzig.de
Reichenbach
Andreas
A
Voigt
Cornelia
C
Boltze
Johannes
J
Bulavina
Larysa
L
Schuhmann
Martin U
MU
Seeger
Johannes
J
Schusser
Gerald F
GF
Freytag
Christiane
C
Grosche
Jens
J
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D001808:Blood Vessels / Q000378:metabolism / Q000473:pathology*; D001921:Brain / Q000473:pathology* / Q000503:physiopathology; D001927:Brain Diseases / Q000473:pathology* / Q000503:physiopathology; D004195:Disease Models, Animal; D017615:Enteric Nervous System / Q000166:cytology / Q000378:metabolism; D005260:Female; D006736:Horses; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence; D009457:Neuroglia / Q000378:metabolism / Q000473:pathology*; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D037121:Plant Lectins; D051381:Rats; D011918:Rats, Inbred SHR; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods
D015415:Biomarkers; D037121:Plant Lectins; C041202:potato lectin