Apotome slider

HT-22 and SH-SY5Y cells were grown on 24-well plates, covered with 13-mm coverslips, and coated with poly-D-lysine (20 μg/mL in PBS-1x) and laminin (5 μg/mL in PBS-1x). After that, the cells were washed with PBS-1x and plated at a seeding density of 6500 cells/well. Then, cells were fixed with 4% paraformaldehyde at room temperature for 10 min and washed 3 × 5 min with 0.1% Triton X-100 in PBS-1x. Next, cells were blocked with donor horse serum 5% in PBST (PBS-Tween 20 0.1%), for 1 h, at room temperature. After that, the primary antibody rabbit polyclonal anti-5-HT3AR) was diluted in donor horse serum 5% in PBST and added to the cells in a ratio of 1:500, following an overnight period of incubation. Then, cells were washed with 0.1% Triton X-100 in PBS-1x (3 × 5 min) and incubated for 1 h at room temperature with the donkey anti-rabbit 488 secondary antibody (1:1000, diluted in donor horse serum 5% in PBST). Finally, cells were washed with 0.1% Triton X-100 in PBS-1x (3 × 5 min), incubated with DAPI (1:1000 in PBS-1x) for 10 min, and washed twice with PBS-1x. For mounting, ProLong^TM gold antifade mountant was used on each slide. Images were acquired on ApoTome Slider (Zeiss^®) fluorescence microscope coupled to the AxioVision Rel. 4.8. software (Zeiss^®).

Images of histological stainings and in situ hybridization were acquired with a slide scanner AxioScan Z1 (Zeiss, Jena, Germany). Fluorescent images of tissue sections were acquired with a Zeiss Axio Imager or Zeiss Axio Observer Z1 equipped with an ApoTome slider for optical sectioning (Zeiss, Jena, Germany). Cleared and whole-mount stained specimen were imaged in ScaleA2 with a light sheet microscope (Lightsheet Z1, Zeiss, Jena, Germany) enabled for dual side illumination and equipped with a 20× detection objective.