Immunocytochemistry Analysis of NF-κB Pathway

ITSCs were seeded on roughened cover glasses with a density of 5 × 10⁴ Cells. Immunocytochemistry was done as previously described in Ruiz-Perera et al. [33 (link)]. Afterwards, Primary antibodies against RELA (mouse, 200,301,065, 1:5000, Rockland, PA. USA), RELB (rabbit, D7D7W, 1:500, Cell Signaling Technology, Danvers, USA), c-Rel (rabbit, 4727, 1:500, Cell Signaling Technology), vGlut-II (rabbit, 07-1402-I, 1:300, Merck Millipore, Burlington, USA), NF200 (mouse, SAB3200747, 1:200, Sigma Aldrich), MAP2 (mouse, sC-390,543, 1:500, Santa Cruz, Dallas, USA) and synaptophysin (rabbit, ab32127, 1:500, Abcam, Cambridge, UK) were incubated for 1 h at RT. Secondary fluorochrome-conjugated antibodies (Alexa 488 anti-rabbit, Alexa 555 anti-mouse, Alexa 555 anti-rabbit, 1:300, Life Technologies, Germany) were incubated for 1 h at RT under the exclusion of light, followed by DAPI nuclear counterstaining. Fluorescence microscopy was performed using the confocal laser scanning microscope LSM 780 (Carl Zeiss, Jena, Germany). Randomly placed pictures were analyzed using ImageJ [34 ]. Nuclear fluorescence intensity was determined using the “measure” function in ImageJ on the nuclei of the cells.