Standardization of formaldehyde-induced fluorescence and its measurement to quantify serotonin emission in pulmonary neuroendocrine cells

Abstract

We describe a modified and standardized quantitative FIF procedure for producing fluorophores and measuring emission intensity of serotonin-containing neuroepithelial bodies (NEB's) in the rabbit lung. This technique, using epifluorescence, was reproduced without significant differences between control groups. Important considerations for reproducibility were: using the same humidity (80% RH) and reaction time (2 h) during the vapor treatment, sectioning at constant relative humidity, avoiding unnecessary heating (sections should not be stretched over a hot plate) and avoiding exposure of sections to light. Optimal emission readings were obtained with sectioning and mounting at 40--50% RH. Readings were reduced by 25% when the mercury light source was switched from 200 W to 100 W. It was also important to let the instruments warm up long enough to avoid drift during quantitation. Each NEB should be subjected to the same duration of light exposure for alignment (30 s) before measuring fluorescence to avoid differences from photodecomposition.