Uplfln10x2ph

Neurons were observed using inverted optical microscopy (IX-71 with $\times$ 10 phase-contrast objective lens, UPLFLN10X2PH, OLYMPUS) equipped with a cooled charge-coupled-device camera imaging system (ORCA-ER, Hamamatsu Photonics).

Extracellular potentials were measured using a self-made 64-channel MEA system as described before [17 (link),18 (link)]. The MEA system was set at a sampling rate of 100 kHz with a low path filter of 2 kHz and a high path filter of 10 Hz, and signals were amplified by 5000 using an analog amplifier. The MEA chips with cardiomyocyte networks were set in the chip holder of the field potential (FP) measurement device on the stage of the inversed optical microscopy (IX-71 with an x10 phase-contrast objective lens, UPLFLN10X2PH, OLYMPUS, Tokyo, Japan) and incubated at 37 °C in 5% CO₂.

For immunocytochemistry, cells were pre-fixed in 2% formaldehyde for 5 min and then fixed in 4% formaldehyde in phosphate buffer (ROTI® Histofix 4%, Carl Roth) for 15 min. After washing with PBS, cells were stored at 4 °C until further use.
To reduce background fluorescence, cells were quenched with 0.1 M NH₄Cl for 10 min at RT. Unspecific binding of antibodies was blocked and cells were permeabilized with normal donkey serum (017-000-121, Jackson ImmunoResearch)/0.3% (v/v) triton (Triton® X-100, Sigma-Aldrich) in PBS 0.01 M for 2 h at RT. Afterward, cells were incubated overnight at 4 °C in PBS 0.01 M/2% bovine serum albumin (001-000-161, Jackson ImmunoResearch)/0.05% sodium azide (S002, Sigma-Aldrich)/0.1% triton with the following primary antibody: rabbit monoclonal anti-NeuN (ab177487, Abcam,1:200). For fluorescence labeling, the following fluorophore-conjugated secondary antibody was used: Alexa Fluor647-conjugated AffiniPure anti-rabbit IgG (H + L) (Jackson ImmunoResearch 1:200).
Images from fixed neurons were taken with a 10 × objective (UPLFLN10X2PH, Olympus) with an epifluorescence microscope (IX81, Olympus) connected to a CCD camera (XM10, Olympus) using the cellSens software (Olympus). Images were subsequently analyzed in Fiji.