Moniliform deformation of retinal ganglion cells by formaldehyde‐based fixatives

Abstract

Protocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate-buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have reported that these fixatives induce the formation of bead-like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage-gated Na + channel toxin, extracellular Ca 2+ ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde-based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry.

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

“…In most experiments, each enucleated eye was hemisected in oxygenated Ames solution at room temperature. For comparison, some eyes were hemisected in ice‐cold, sucrose‐supplemented phosphate buffer (PB) (Stradleigh et al, ). After removal of the vitreous, the resulting eyecups were processed as either whole mounts or vertical sections.…”

“…To form flat mounts, the retina was isolated with forceps, placed photoreceptor‐side down on a membrane filter (PICM0RG50, Millicell; Millipore, Tuliagreen, Ireland), and immersed in a prefixative conditioning solution containing sucrose (200 mM) for an additional 30 min (Stradleigh et al, ). Although this solution was ice‐cold in most experiments, similar results were obtained when it was room temperature or even warmed to 37 °C.…”

“…Although this solution was ice‐cold in most experiments, similar results were obtained when it was room temperature or even warmed to 37 °C. Membrane‐attached retinae were then fixed either overnight at 4 °C, or for 20 min at room temperature, in a fixative containing formaldehyde (4% w/v) and sucrose (200 mM) in 100 mM 4‐(2‐hydroxyethyl)‐1‐piperazine‐ethanesulfonic acid (HEPES) (Stradleigh et al, ; cf. Stell & Lightfoot, ; Anderson et al, ; Pallotto, Watkins, Fubara, Singer, & Briggman, ).…”

“…It is possible that TH cells release dopamine by different mechanisms in the inner and outer plexiform layers, and that TH cell shape and ion channel distribution vary with species. However, most studies to date have examined TH cells in retinae that were incubated in aldehyde‐based fixatives that we have found to deform, and reduce the detectability of, retinal ganglion cell axons and dendrites (Stradleigh, Greenberg, Partida, Pham, & Ishida, ). The present study examines the shape, contour, and immunostaining of TH cells by use of protocols we developed to preserve the dendritic and axonal morphology of proximal retinal neurons and to localize transmembrane and membrane‐associated proteins (Stradleigh et al, ).…”

Evidence for an enduring ischaemic penumbra following central retinal artery occlusion, with implications for fibrinolytic therapy

“…Of note, fusiform varicosities rich in mitochondria have been reported to be a normal feature of RGC axons in humans and non-human primates, especially in the retinal periphery (Wang et al, 2003). However, such axonal beading isn't seen on in vivo imaging (Gray et al, 2008), and is almost certainly a fixation artefact (Stradleigh et al, 2015).…”