Figure 1. 1000-fold reduction of conventional FG-concentrations leads to intact FG-labelled DA neurons in vivo.

(A) Confocal images of a dilution series of Fluorogold/ACSF injections. (Top panel, 1^st row) merged images of injections sites (4x magnification) in dorsal striatum with TH in green and FG in blue. (2^nd and 3^rd row) 10x images of retrogradely traced neurons (FG black in the 2^nd row and blue in the 3^rd row) in the midbrain (TH green) at bregma −3.16 mm (10x magnification). (Bottom panel) Images of retrogradely traced SN DA neurons at higher magnification (60x; FG black in the 1^st row and blue in the 3^rd row, TH green). Note that 0.002% FG constitutes the lowest detectable concentration. (B) Use of FG-Antibody facilitates identification of FG-labelled neurons after 0.002% FG/ACSF infusion. Note the increased detectability in comparison to the intrinsic FG signal after 0.002% infusion of FG (Figure 1A, column four).

Figure 1—figure supplement 1. FG-labelling detection method.

(A) Detection method of intrinsic FG signals. The left image shows the TH-mask (TH in green), the right image shows the FG-channel (FG in black), the TH-mask is overlaid on both images in magenta, the four intensity histograms depict the background and single neuron FG-intensity distributions with a dotted line at the threshold of labelling detection. Threshold for detection was set to Thr = mean background intensity + 2*SD of the mean background. TH-negative nuclei were subtracted from the background. (B) Detection method of immune-FG-signal. The left image shows the TH-mask (TH in green), the middle image shows the FG-channel (FG in black), the intensity histogram displays the FG-intensity distribution of the TH-free background, the right image shows the thresholded FG-channel, the TH-mask is overlaid on all images in magenta. Here, threshold for detection was set to Thr = mean background intensity + 10*SD of the mean background.