Streptavidin alexa fluor 568 conjugate

A fluorescent labeling with maackia amurensis agglutinin (MAA) and sambucus nigra amurensis (SNA) lectins was also performed as described above except that the digoxigenin‐labeled lectin was used at 50 µg·mL⁻¹ (for MAA) and at 5 µg·mL⁻¹ (for SNA) and that it was detected with DyLight 488 Anti‐DIG (green for MAA; 1 : 1000; Vector Laboratories, Burlingame, CA, USA) and Streptavidin Alexa Fluor 568 conjugate (red for SNA; 1 : 200; Invitrogen, Carlsbad, CA, USA).

Electrodes were filled with intracellular solution supplemented with 0.2%–1% biocytin. In this solution, KMeSO₄ was reduced to 119.5 mm to maintain osmotic balance. After the experiment was completed, the electrode was left in place for a minimum of 20 min to allow for adequate filling. Slices were then fixed in 4% paraformaldehyde and refrigerated overnight, washed in PBS, and incubated overnight in streptavidin Alexa Fluor 568 conjugate (1:1000, Invitrogen) at 4°C with agitation. Slices were then washed three times in PBS and mounted. Filled neurons were imaged with an Olympus FluoView laser scanning confocal microscope equipped with a krypton laser and a 20× objective. To capture the full extent of the dendritic tree, most neurons were imaged in two partly overlapping stacks of equal depth. These stacks were stitched together using the “stack combiner” plug-in (MacBiophotonics) within ImageJ (NIH). More than 40 rat cells were filled and imaged, but only 11 of these yielded adequate morphologic reconstruction and the complete set of electrophysiological measurements necessary for analysis. Confocal image stacks from these 11 neurons were used to reconstruct the cells in 3D using Neurolucida (MBF Biosciences). The 3D reconstruction was then analyzed using Neuroexplorer (MBF Biosciences).