Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

GFP nanobodies reveal recently-exocytosed pHluorin molecules.

Neurotransmitter release requires vesicle recycling, which consists of exocytosis, endocytosis and the reformation of new fusion-competent vesicles. One poorly understood aspect in this cycle is the fate of the vesicle proteins after exocytosis, when they are left on the plasma membrane. Such proteins are often visualized by coupling to pH-sensitive GFP moieties (pHluorins). However, pHluorin imaging is typically limited by diffraction to spots several-fold larger than the vesicles. Here we show that pHuorin-tagged vesicle proteins can be easily detected using single-domain antibodies (nanobodies) raised against GFP. By coupling the nanobodies to chemical fluorophores that were optimal for super-resolution imaging, we could analyze the size and intensity of the groups of pHluorin-tagged proteins under a variety of conditions, in a fashion that would have been impossible based solely on the pHluorin fluorescence. We conclude that nanobody-based pHluorin detection is a promising tool for investigating post-exocytosis events in neurons.

Optical dissection of experience-dependent pre- and postsynaptic plasticity in the Drosophila brain.

Drosophila represents a key model organism for dissecting neuronal circuits that underlie innate and adaptive behavior. However, this task is limited by a lack of tools to monitor physiological parameters of spatially distributed, central synapses in identified neurons. We generated transgenic fly strains that express functional fluorescent reporters targeted to either pre- or postsynaptic compartments. Presynaptic Ca(2+) dynamics are monitored using synaptophysin-coupled GCaMP3, synaptic transmission is monitored using red fluorescent synaptophysin-pHTomato, and postsynaptic Ca(2+) dynamics are visualized using GCaMP3 fused with the postsynaptic matrix protein, dHomer. Using two-photon in vivo imaging of olfactory projection neurons, odor-evoked activity across populations of synapses is visualized in the antennal lobe and the mushroom body calyx. Prolonged odor exposure causes odor-specific and differential experience-dependent changes in pre- and postsynaptic activity at both levels of olfactory processing. The approach advances the physiological analysis of synaptic connections across defined groups of neurons in intact Drosophila.