Confocal lsm510 meta microscope

Single-cell Ca²⁺ levels in cortical neurons transduced with GFP or GFP-PKD1-Ca lentiviral particles were recorded using the ratiometric Ca²⁺ indicator dye Fura Red acetoxymethyl ester (ThermoFisher Scientific). Cells were grown on eight-well chamber slides and loaded with 5 μM Fura Red, AM for 30 min at 37 °C in HBSS containing 145 mM NaCl, 5 mM KCl, 0.75 mM Na₂HPO⁴, 10 mM glucose, 10 mM HEPES (pH 7.4), 1 mM MgCl₂, and 2 mM CaCl₂ (high Ca²⁺ medium), and then rinsed and left undisturbed for 30 min at 37 °C to allow for de-esterification. Measurements of intracellular Ca²⁺ levels were performed every second at 37 °C using a Confocal LSM510 META microscope (Zeiss, Germany). Changes in intracellular Ca²⁺ concentration ([Ca²⁺]i) in individual neuronal cell bodies are expressed as the F458/F488 ratio after subtracting background fluorescence. This ratio represents the emission intensities at 660 nm obtained after excitation at 458 and 488 nm. Cells that responded rapidly to NMDA stimulation were identified as neurons and only GFP+ neurons were analyzed. Data processing was performed using ImageJ software.

The cartilage samples were fixed in 4% paraformaldehyde (PFA) for 1 h and then stored in 35% ethanol at 4 °C and then dehydrated, cleared in Histoclear, and embedded in paraffin. The tissue block was sectioned to achieve both transverse and cross-section configuration on the slide and stained with H&E and Hoechst stain. Hoechst stain (1 µg/mL) was placed on the sample for 5 min, then rinsed 3 times with PBS. The samples were then imaged on a Zeiss Confocal LSM 510 Meta microscope.

MEFs, transfected with the four p38 variants, were grown on sterile coverslips in 6-well plates. Cells were washed with PBS with 100 mg/L calcium chloride and 100 mg/L magnesium chloride three times, fixed with 4% paraformaldehyde for 15 min, and washed again with PBS three times. Next, the cells were perforated with 0.1% Triton X100 for 30 min. After washing the cells three times with PBS, they were stained with 3.5 μM Phalloidin Conjugates TRITC (Sigma, St. Louis, MO, USA) and 1:1000 Hoechst for 60 min. The slides were stored in PBS at 4 °C until use. The confocal images were taken by Confocal LSM 510 META Microscope (Zeiss, Oberkochen, Germany).