Immunochemical or fluorescent labeling of vesicular subcellular fractions for microscopy imaging.
We describe a procedure for the labeling of membranous vesicular purified subcellular fractions, to image them, typically by confocal laser scanning microscopy. Being intracellular organelles, these fractions, once purified cannot be attached to glass slides as for cells. Fractions are labeled "in batch" without prior embedding or freezing. Each labeling step performed by passages of resuspension/centrifugation is followed by washings. Then samples are dispersed on the glass slides. Mammalian retinal rod outer segment disks, intact brain stem myelin vesicles, and brain synaptosomes were chosen, as these subcellular fractions can be purified by well established procedures. These fractions were immunolabeled with specific antibodies. Moreover, by the earlier procedure, we show that the mitochondrial vital membrane potential probe MitoTracker Deep Red 633 stains myelin vesicles and rod disks before fixation, consistently with our previous reports of a respiring capacity of these membranes. Therefore, the technique seems adequate to become an instrument to study the structure and the function of these and other subcellular fractions.
Department of Biology, University of Genoa, Italy. Isabella.Panfoli@unige.it
Isabella
I
Panfoli
Daniela
D
Calzia
Silvia
S
Ravera
Paolo
P
Bianchini
Alberto
A
Diaspro
D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007120:Immunochemistry / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D009186:Myelin Sheath / Q000737:chemistry; D017072:Neostriatum / Q000166:cytology; D016548:Prosencephalon / Q000166:cytology; D012374:Rod Cell Outer Segment / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D013347:Subcellular Fractions / Q000737:chemistry*; D013574:Synaptosomes / Q000737:chemistry
D002232:Carbocyanines; D005456:Fluorescent Dyes; C089187:cyanine dye 3