Donkey anti rabbit alexa 594

Cells at 2 DIV were fixed for 20 min at room temperature in 4% paraformaldehyde and permeabilized for 4 min with 0.12% Triton-X plus 0.12% gelatin in phosphate buffered saline (PBS). Cells were then washed with PBS/gelatin and incubated for 1 h with chicken anti-microtubule associated protein-2 (MAP2) (Abcam, Cat# ab75713, diluted 1:10.000 in PBS/gelatin) and rabbit anti-TAU (Abcam, Cat# ab47568, diluted 1:500 in PBS/gelatin) or goat anti-GFP (Abcam, Cat# ab5450, diluted 1:1000 in PBS/gelatine) to enhance the fluorescence of the transfected cells. After washing in the same buffer, cells were incubated for 1 h at room temperature with Alexa fluor donkey anti chicken (Jackson Immunoresearch, Cat# 703-545-155) and Alexa 594-donkey anti- rabbit (Jackson Immunoresearch, Cat# 711-585-152 diluted 1:1000 in PBS/gelatin). Cell nuclei were stained with DAPI.

To assess overall neuronal activity of the DRN immunohistochemical expression of c-Fos was evaluated. The DRN sections were incubated overnight with a primary anti-c-Fos antibody (rabbit polyclonal anti-c-Fos; Abcam; 1:1000). This was followed by incubation for one hour with a secondary antibody (donkey anti-rabbit alexa 594, Jackson immunoresearch Laboratory; 1:200). Eight slices were selected from bregma − 4.16 to − 4.96 and photographed with an Olympus BX51 fluorescence microscope (Olympus, Germany) connected to an Olympus Camera DP72 (Olympus, Germany). All clear c-Fos expressing neurons were counted (FiJi v2.0.0, National Institutes of Health; Maryland).