Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

Abstract

For ultrastructural studies, it is of great interest to be able to combine anatomical tracer techniques with sensitive immunohistochemical methods. Fluorogold (FG) is a fluorescent and retrogradely transported anatomical tracer, which is commonly used to label neurons in the brain and spinal cord for light microscopic studies. We here describe a method for detecting FG-labeled somata in the electron microscope using a high resolution post-embedding immuno-gold method. For this purpose, spinal motoneurons were retrogradely labeled by an intraperitoneal injection of FG in the adult rat. The rats were intravascularly perfused with a fixative solution containing 2% paraformaldehyde and 1-2% glutaraldehyde. Vibratome sections of spinal cord tissues were cryo-protected in glycerol, freeze-substituted in methanol containing uranyl acetate, and embedded in the Lowicryl HM20 resin at low temperatures. Electron microscopic analysis demonstrated atypical lysosome-like structures in the cytoplasm of FG-labeled motoneurons. Subsequent post-embedding immuno-gold labeling demonstrated prominent accumulation of FG in numerous lysosomes but not in other organelles or cytoplasmic compartments of the labeled neurons. The protocol is versatile and allows for combining anatomical tracing of neurons with e.g. neuro-transmitter studies in the electron microscope. We suggest that the described method for sensitive detection of FG in the spinal cord may also have broad applicability to other areas of the central nervous system.

Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture

“…The grids were rinsed and blocked with 1:20 dilution of normal goat serum (Pierce) in Tris-buffered saline and then incubated with two different antibodies in one of the following combinations sequentially: 1) Anti-CD63(LAMP3) and anti-LAMP1, 2) Anti-Rab5 and anti-Rab7, each at 1:100 dilutions. The grids were washed with Tris-buffered saline thoroughly and incubated with appropriate secondary antibody tagged to either 12-nm or 18-nm gold particles (1:200 dilution) following a standard protocol (34).…”

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

“…The blocked grids were incubated with two different antibodies in one of the following combinations (the antibody incubations were done consecutively, each incubation period was for 24 h): 1) anti-␣A and anti-␣B, 2) anti-␣A and antiRibophorin-1, and 3) anti-␣B and anti-Ribophorin-1, with each at 1:200 dilutions. The grids were washed with Tris-buffered saline containing 0.001% Triton X-100 thoroughly and incubated with secondary antibodies tagged to either 12-nm or 18-nm gold particle (1:300 dilution) following a standard protocol (21).…”

A simple method to fluorescently label pericytes in the CNS and skeletal muscle

“…IP administration of FG results in labelling of CNS pericytes within 3 hours, with a punctate intracellular pattern suggesting it is localised to lysosomes as it is in neurones (Persson and Havton, 2009). FG labelling of pericytes can still be observed up to 48 hours post i.p.…”