4.4.4. Confocal Co-Localization and Particle Analysis

The CHO-K1 cells transfected with K8 or K18 and p62-H1, p62-H2, or p62∆SH2 were fixed and immunolabeled. High-resolution images of cells were recorded using a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100× objective lens (Plan-Fluor ×100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, an AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems), and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric corporation). AF488 and rhodamine were alternately excited with 488 and 561 nm laser lines, respectively, and emissions were acquired at 353 and 600 nm using a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). The Z-stacks of both channels in 0.2 μm increments were recorded. VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. For IHBs, hybrid inclusions and MDB tissues, LSM 510 META (Carl Zeiss, Jena, Germany) confocal laser scanning microscope with 458 nm, 590 nm, and 694 nm filters were used. Images were blind deconvoluted with NIS-elements (Nikon, Austria). The co-localization was determined on a single cell level for cell culture experiments and carried out on the whole image for tissue experiments using ImageJ and the plugin coloc2. The Pearson correlation coefficient was chosen for quantitative comparison. The size of the AF488 and rhodamine-labeled structures were determined using a custom-made ImageJ macro. After using the rolling ball background correction, the Z-stacks were binarized with a combination of global stack comprehending Otsu auto threshold and a local Otsu auto threshold with an x/y-radius of 10 pixels. The binary Z-stacks were further analyzed using the 3D manager plugin to determine for each cell the count, volume, and surface of AF488 and rhodamine-labeled particles. The particle size of p62 (p62CT and p62NT)-labeled structures in tissue samples was measured with the particle analyzer in ImageJ after background subtraction using the rolling ball method and Otsu auto thresholding for binarization.