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Preparations were fixed with Quick Dip Fixative (Fronine Australia) and stained with Quick Dip (Fronine).
When antibodies against any of the glutamate receptor subunits were used, preparations were fixed 30 min in Bouin's fixative.
For these stainings, preparations were fixed for 15 min. in Bouin's fixative (Sigma, L'isle d'Abeau, France).
All cell preparations were fixed with Cytofix (BD Biosciences).
Single cell preparations were fixed in 4% paraformaldehyde in 0.1 M PBS for 20 minutes.
Cells were washed and all cell preparations were fixed with Cytofix (BD Biosciences).
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Note that the Sec8 antibody recently described in [ 21] as not showing immunoreactivity at the fly NMJ, actually does so (and shows staining very similar to our antibody) when preparations are fixed in Bouin's fixative.
The intact head neck preparation was fixed at the thoracic end with the head unconstrained.
To compare fluorescence signals from different preparations, settings were fixed for all samples within the same analysis.
For immunohistochemistry preparation, brains were fixed in formalin (4% formaldehyde in 1× PBS, pH7.4) for 24 hours and then embedded in paraffin.
For histological preparation samples were fixed for 24 hr at 4°C, then centrifuged again (3 min, 0.84 × g) and pellets were embedded in Histogel® (Richard-Allan Scientific, Microm International, Walldorf, Germany; as specified by the manufacturer) and subsequently in Paraplast® (Vogel) by means of an automatic embedding device.
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