Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy

• Published in: The journal of histochemistry and cytochemistry Vol. 41; no. 8; pp. 1267 - 1272
• Main Authors: Hulspas, R, Krijtenburg, PJ, Keij, JF, Bauman, JG
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA Histochemical Soc 01.08.1993; SAGE Publications; Histochemical Society
• Subjects: Avidin; Biological and medical sciences; Diverse techniques; Eosine Yellowish-(YS) - analogs & derivatives; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Lasers; Microscopy, Fluorescence - methods; Molecular and cellular biology; Immunohistochemistry; Fluorescent tracer; Bleaching; Emission; Fluorescence; Excitation; Method
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1177/41.8.7687265

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.