Digital analysis of light microscope immunofluorescence: high-resolution co-localization of synaptic proteins in cultured neurons

• Published in: Journal of neuroscience methods 2000-03, Vol.96 (1), p.1-9
• Main Authors: Hutcheon, B, Brown, L.A, Poulter, M.O
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Biological and medical sciences; Cerebral Cortex - chemistry; Cerebral Cortex - cytology; Cortex; Deconvolution; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; GABA A-receptor; General aspects. Models. Methods; Image Processing, Computer-Assisted - methods; Imaging; Microscopy, Fluorescence - methods; Microspheres; Neurons - chemistry; Rats; Rats, Sprague-Dawley; Receptors, GABA-A - analysis; Receptors, GABA-A - immunology; Synapse; Synapses - chemistry; Vertebrates: nervous system and sense organs
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/S0165-0270(99)00148-X

A protocol is presented for determining the subcellular distribution of fluorescently labeled proteins in neurons using deconvolved images gathered with a wide-field microscope. The protocol includes optimal settings for the numerical algorithm used to deconvolve the images and an objective method for thresholding the deconvolved images to retain only high-intensity, specific labeling. The effectiveness of the protocol is demonstrated using a fluorescent antibody stain directed towards the α1 subunit of the GABA A receptor in cultured neurons. We also show, using an antibody against the presynaptic vesicular protein synaptophysin, that the technique can detect presumptive regions of synaptic contact between neurons. Double-labeling with the anti-α1 and anti-synaptophysin antibodies in a cultured neuron reveals regions of both synaptic and non-synaptic α1 labeling. Thus, numerical postprocessing of wide-field images can be used to efficiently locate receptor proteins in neurons in relation to functionally important structures. This confocal-like functionality is attained without the excessive bleaching and phototoxicity associated with the intense laser excitation light used in confocal techniques.