Quantifying Neuroblastoma/Glioma Cell Responses

Neuroblastoma/glioma cells at 4 × 10³ were seeded on glass coverslips in a 12-well plate for 24 h. Treatments with the indicated DMSO, CAPE, and RA concentrations were given for indicated time points followed by the fixation with pre-chilled absolute methanol at reverse transcriptase (RT) for 10 min. Fixed cells were subsequently permeablized with phosphate-buffered saline (PBS)-Triton-X-100 (0.2%) for 10 min followed by blocking with 2% bovine serum albumin (BSA) for 20 min. Indicated primary antibodies (please see the details of used antibodies in Supplementary Table 1) were incubated on RT for 1 h or at 4°C overnight. Cells were subsequently incubated with Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) and counterstained with Hoechst 33258 (Roche, Basel, Switzerland) as described earlier (Kalra et al., 2018 (link)). Immunofluorescence images were acquired under a Carl Zeiss Axioplan-2 microscope and captured with a Zeiss AxioCam HRc camera. The intensity of the acquired immunofluorescence images was quantitated by the ImageJ software that was further normalized with the respective controls and represented as % change over control.