Alexa fluor 546 goat anti mouse igg

Abundance of Bax and Bcl-xL was assessed as previously described^{5 (link)}. Briefly, 3 days old PTC were incubated with control or HG for 8 h. Cells were fixed with 4% paraformaldehyde (pH 7.4) for 10 min, permeabilized with 0.3% Triton X-100 for 10 min and blocked with 5% BSA in 0.1% Triton X-100 for 1 h. Primary antibodies mouse monoclonal anti-Bax (6A7) (5 μg/ml) (Abcam, Cambridge, UK) and rabbit monoclonal anti-Bcl-x_L (54H6) (1:200) (Cell Signaling Technology, Danvers, MA, USA) were applied over night at 4 °C. Cells were washed and secondary antibodies Alexa Fluor 546 goat anti-mouse IgG (Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 546 goat anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA) were applied for 1 h at room temperature. Secondary antibody controls were subjected to the same treatment, but primary antibodies were omitted. Cells were imaged with a Zeiss LSM 510 confocal microscope equipped with × 63/1.4 NA oil objective. The microscope setting was kept fixed for all measurements. The Bax and Bcl-xL abundances were analyzed in Matlab (The MathWorks, Natick, MA, USA). The total abundance of Bax and Bcl-xL was calculated as the percentage of Bax or Bcl-xL (pixels) normalized to cell size (pixels). On each coverslip, at least three cells were analyzed. The control group was set to 100%.

Sections were cut on a freezing microtome (40 µm thickness) and were rinsed 3 times (5 min each) in PBS, then permeabilized with 3 washes (5 min each) in PBS with 0.2% Triton X-100 (Sigma-Aldrich). Sections were then quenched with 0.1 M glycine for 20 min. After that, they were placed in 10% normal goat serum (NGS, Thermoscientific) in PBS at room temperature (RT) for 1 h to block nonspecific antigens. Sections were then incubated in primary antibodies overnight at 4 °C in darkness with both chicken anti-tyrosine hydroxylase (TH) antibody (1:500, Abcam) and mouse anti-mCherry antibody (1:200, Ozyme) in PBS containing 0.1% Triton X-100 and 3% NGS. After three 5 min rinses in PBS, sections were then incubated with secondary antibodies in PBS containing 3% NGS for 1 h at RT in darkness. Secondary antibodies used in this study were Alexa Fluor 488 goat anti-chicken IgG (1:3000, Life Technologies) and Alexa Fluor 546 goat anti-mouse IgG (1:3000, Life Technologies). Then slices were washed three times (5 min each) in PBS, mounted onto slides (Superfrost) and coverslipped with fluorescence mounting medium (DAKO).