Journal: Scientific Reports
Article Title: Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABAA receptor clustering induced by inhibitory synaptic plasticity
Figure Lengend Snippet: Correlative light-high resolution scanning electron microscopy (CL-HRSEM) localization of GABA A Rα1 in control (CTRL, a–e) and iLTP (NMDA, f–i) primary hippocampal neurons growing on photo-etched coverslips. The BSE signal (pseudo-coloured in yellow) is superimposed on the grey-scale SE images. ( a ): low magnification HRSEM images of a CTRL neuron immunolabelled for GABA A Rα1. Inset: same neurons imaged by CFM. ( b ): CFM image of part of the neurite bundle boxed in a. Note the presence of GABA A Rα1 clusters (bright spots) along the neurite. The boxed regions are magnified in c (insets) and in d–e. ( c ): HRSEM image showing the same region boxed in a. The yellow spots on the neurite bundle are GABA A Rα1 receptor clusters. The double inset shows higher magnifications of the single neurite boxed in (c) (bottom left) imaged respectively at the CFM (above) and at the HRSEM (below). The arrowheads point to neurites without gold nanoparticles; ( d ): HRSEM image of the bundle of neurites boxed in c (upper right); ( e ): CFM image of the same bundle of neurites imaged in d. The arrow points to a fluorescent spot not observed in d; ( f ): low magnification HRSEM images of a NMDA stimulated neurons immunolabelled for GABA A Rα1. Inset: the same neurons imaged by CFM; ( g ): HRSEM image showing a portion of the cell body of the neuron imaged in f (upper Inset). Left inset: the same region imaged at the CFM. Right Inset: HRSEM higher magnification of the region boxed in g. ( h ): HRSEM image of the region boxed in f (bottom Inset). The arrowheads point to neurites without gold nanoparticles; ( i ), CFM image of the same region imaged in h. The arrow points to a fluorescent spot not observed in h. Circles and brackets point to the same sub-regions. Scale bars are 10 µm in a, a inset, f, f inset; 5 µm in b and g right inset; 1 µm in c–e and g–i; 0.2 µm in g right inset.
Article Snippet: During the recovery period, 12 minutes after the end of the NMDA treatment, live immunolabelling of GABAA Rα1 subunits was performed, namely cells were incubated 13 minutes in a solution containing primary antibody against GABAA Rα1 (Alomone Labs, Israel) diluted 1:30 in 0.5% bovine serum albumin (BSA), 350 mM sucrose in PBS, followed by 13 minutes incubation with gold-conjugated secondary antibody.
Techniques: Electron Microscopy