Axiovert 100 m confocal microscope

To determine the anatomical contribution of AS (Group 2) and PS (Group 4) of the straight heart tube to development from the U loop (HH16) through the tetracavitary heart (HH34), the embryos were labeled and cultured in ovo. In order to place the label, a window, measuring approximately 1 cm², was opened in the egg shell. After exposing the heart (HH16) by dissecting the vitelline and pericardial membranes, a small piece (0.5 mm) of the previously prepared glass filament embedded in Dil was adhered to the myocardium in the areas at which the tracked labels were located in the in vitro analysis. In both groups (G2 and G4), the shell window was covered with an adhesive tape, and the eggs were incubated at 37°C undersaturated humidity until development of the tetracavitary mature heart (HH34). The final position of the fluorescent labels was determined as described in the in vitro culture. In contrast, to identify the position of the fluorescent labels in the internal structures of the heart, eight fixed mature hearts were dehydrated and embedded in PEG1400, as described by Lazik et al. (1997) [38 ]. Histological sections (8μm) of the heart were obtained and observed under an Axiovert 100M confocal microscope (Carl Zeiss).

We used BacMam LC3B-GFP as a marker for autophagy. Once differentiated on coverslips for 7 days, control cells and cells treated with different concentrations of ATX were transfected with BacMam LC3B-GFP or BacMam LC3B (G120A)-GFP viral particles (MOI = 30) for 18–20 h, according to the Premo Autophagy Sensor Kit. LC3B-GFP and LC3B (G120A)-GFP signals were monitored and captured using a Zeiss Axiovert 100M confocal microscope.

DA neurons were fixed for 15 min in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS at room temperature and then incubated in blocking buffer (1X PBS, 1% Goat serum, 4% BSA) for 1 hr. Cells were permeabilized in 0.2% Triton X-100 in PBS for 10 minutes and incubated overnight with primary antibodies. Primary antibodies are listed in the Supplemental Methods. The Click-iT EdU Alexa Fluor^® 488 Imaging Kit was used to assess the mitotic state of TH⁺ neurons. Confocal and spinning disk epifluorescence confocal imaging analysis was performed using a Zeiss LSM700 or Olympus Axiovert 100 M confocal microscope (Carl Zeiss, Olympus). Cells were counted using Zeiss confocal microscope software (Zeiss). Ten images from one coverslip from two independent experiments were used for counting.