Mcherry antibody

The viral expression in the injected mice was immunohistologically validated. Mice were transcardially perfused with PBS, pH 7.4. The brains were removed and fixed overnight in 4% PFA. One hundred μm of free-floating slices were permeabilized in 0.5% Triton X-100 for 1 hr. Subsequently, slices were incubated with a mCherry antibody (1:10,000, rat serum, Thermo Fisher Scientific, M11217) in a blocking reagent (4% normal goat serum, 0.4% Triton 1%, and 4% BSA in PBS) over night at room temperature. After washing the samples three times with PBS, a secondary antibody was administered (Alexa Fluor 594 goat anti-rat, 1:400, Invitrogen, A11007) in 5% normal goat serum/BSA and incubated for 2 hr at room temperature. During the last 30 min of incubation, Nissl staining was carried out (Invitrogen, N21473, 1:200). Afterward slices were washed three times with PBS, mounted with Dako Mounting Medium, and covered with a glass cover-slip. Alternatively, a NeuN staining was performed to visualize neurons in the hippocampus using a NeuN primary antibody (1:1000, MABN140, rabbit, Sigma-Aldrich, secondary Alexa Fluor 405 goat anti-rabbit, A31556, 1:400on).

To look at the localisation of NGF in undifferentiated cells, 48-h post-transfection, PC12 cells were fixed in 4% paraformaldehyde for 10 min. To look at NGF localisation in differentiated cells, 24 h post-transfection cells were treated with 100 ng/ml mouse NGF (Peprotech) for five days and then fixed. Cells were stained with an mCherry antibody (ThermoFisher Scientific 16D7, 1:1000), and Alexa-546 conjugated secondary antibodies were purchased from Invitrogen (1:1000).
All images were acquired with an LSM710 laser-scanning Meta confocal microscope (Carl Zeiss) using a ×63 oil-immersion objective.