Axio observed z1

MitoPY1 was used to measure mitochondrial-derived H₂O₂ in primary hippocampal neurons. Cells were incubated with 10 µM MitoPY1 in Na⁺ medium at 37 °C for 30 min. Then, cells were washed to remove the not-internalised probe, and changes in mitochondrial H₂O₂ levels were analysed in Mg²⁺-free Na⁺ medium plus serine/glycine using confocal images obtained using a 20× objective with NA = 0.8 on a Carl Zeiss Axio Observed Z1 inverted confocal microscope using the CSU-X1M spinning disc technology (Zeiss, Jena, Germany). Basal mitochondrial H₂O₂ levels were recorded for 15 min basal followed by 30 min after stimuli (one frame every minute). Fluorescence intensity was quantified using FIJI software (version 2.1.0/1.51w).

Mitochondrial morphology was assessed as in [24 (link)]. Briefly, hippocampal neurons, cotransfected with MitoDsRed plus GFP, were washed and incubated in Na⁺ medium plus serine/glycine at 37 °C for mitochondrial movement studies. An image of neuronal projections (MitoDSRed plus GFP) was acquired using a 63× objective with NA = 1.4 on a Carl Zeiss Axio Observed Z1 inverted confocal microscope using the CSU-X1M spinning disc technology (Zeiss, Jena, Germany) after 10 min incubation with AβO in the absence or presence of SU6656 or MK-801. To assess mitochondrial morphology, the macros AutoROI and MitoProtAnalyser for FIJI were applied in the last image acquired to assess the following parameters per mitochondria: aspect ratio (major axis/minor axis) and circularity (4π × area/perimeter²), which reflect length and degree of fragmentation, respectively.

Cells were grown in X-well Tissue Culture Chambers (Sarstedt). Effect of treatment were stopped by washing with ice-cold PBS, and cells were fixed by incubation inn methanol for 4 min, before incubation overnight with antibodies against p65, AhR or Arnt (working dilution 1: 200 in PBS with 1% BSA). After washing and incubating for 3 h with secondary antibodies conjugated with Alexa Fluor®488 or 594, the preparations were visualized using a Zeiss AxioObserved.Z1 equiped with an AxioCam ERc 5 s digital camera (Zeiss).