Two-color fluorescence labeling in acrolein-fixed brain tissue.
Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-mum sections from acrolein-perfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling.
Luquin
Esther
E
Center for Applied Medical Research (CIMA), Area de Neurociencias, Universidad de Navarra, Pamplona, Spain.
Pérez-Lorenzo
Eva
E
Aymerich
María S
MS
Mengual
Elisa
E
D000171:Acrolein; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001894:Borohydrides; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D005404:Fixatives; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide; D007202:Indicators and Reagents; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D051381:Rats; D017208:Rats, Wistar
D015415:Biomarkers; D001894:Borohydrides; D005404:Fixatives; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D000171:Acrolein; C025364:sodium borohydride; D006861:Hydrogen Peroxide