4.6.1. Detection of NF-κB p65 Subunit Nuclear Translocation

The NF-κB pathway activation (p65 nuclear translocation) in HCPEpiC cultures was visualized via p65 nuclear immunofluorescence staining based on the method drawn from our previous study [38] with some minor modifications. For this purpose, HCPEpiCs were cultured in BioLite 24-well plates (Thermo Scientific) on sterile glass microscope slides at a density of 5 × 10⁴ cells/slide. Cells were then treated with IVH-III, IVH-IV, or non-IVH control CSF samples (10 v/v %) for 1 h. After treatment, cells were washed twice with DPBS solution and fixed with ice-cold methanol-acetone (50 v/v %, Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Non-specific antibody binding sites were blocked with FBS (Sigma-Aldrich) for 15 min. For primary labeling of the NF-κB p65 subunit, a polyclonal rabbit anti-human p65 antibody (100 μg/mL, sc-372, Santa Cruz Biotechnology, Dallas, TX, USA) was used for 1 h, followed by secondary staining with Alexa Fluor 488-conjugated goat-anti-rabbit IgG (5 μg/mL, A32731, Invitrogen, Carlsbad, CA, USA) for 1 h. Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) and samples were observed by Zeiss Axio Scope A1 fluorescent microscope (HBO 100 lamp) (Carl Zeiss Microimaging GmbH, Goettingen, Germany); DAPI: excitation at 365 nm and emission BP filter 445/50 nm; and fluorescein: excitation of BP filter at 470/40 nm and emission BP filter 525/50 nm. Images were analyzed with ZEN 2012 v.1.1.0.0. software (Carl Zeiss Microimaging GmbH). The ratio of nuclear and cytosol fluorescence intensity was calculated for NF-κB p65 staining. The specificity of immunostaining was checked by incubating the cells with the secondary antibody alone and only a limited background staining was seen.