2.8. Rat Cortical Phospho-CREB Immunocytochemistry

Rat cortical cells were treated either with MQ or ARC39 10 µM (ARC39 was solved in MQ) concomitantly with DMSO or hyperforin 1 µM (hyperforin was solved in DMSO) 24 h prior to fixation. ARC39 is a synthetic ASM inhibitor, which directly inhibits ASM’s catalytic activity [45,46].

Cells on cover slips were fixed with 4% (w/v) paraformaldehyde for 3 min and afterwards blocked (10% (v/v) FCS, 0.1% (w/v) glycine in PBS 1x) for 45 min. Primary antibody solution was prepared in staining solution (3% (v/v) FCS in PBS 1x): MAP 2 ms (Sigma #M4403, 1:1000), GAD 2/65 gp (Synaptic Systems #198104, 1:500; Synaptic Systems GmbH, Göttingen, Germany), pCREB rb (Ser133) (CST #9198, 1:1000; Cell Signaling Technology, Danvers, MA, USA). MAP 2 was used to stain exclusively neuronal cells. GAD 2/65 was used to distinguish between excitatory and inhibitory neurons, only cells which were negative for GAD 2/65 were taken into consideration to analyze exclusively excitatory neurons. Cover slips were incubated with primary antibody overnight at 4 °C. The next day, cover slips were washed with PBS 1x. Secondary antibody solution was prepared in staining solution: anti-mouse donkey Cy5 (Jackson ImmunoResearch #715-175-150, 1:1000; Jackson ImmunoResearch Europe Ltd., Ely, UK), anti-guinea pig donkey alexa 488 (Jackson ImmunoResearch #706-545-148, 1:1000), anti-rabbit donkey Cy3 (Jackson ImmunoResearch #711-165-152, 1:1000). Cover slips were incubated with secondary antibody for 1 h at RT. Cells were washed with PBS 1x and with MQ and finally mounted on glass slides with Fluoroshield mounting media, including DAPI (Sigma #F6057). Imaging was performed at a Nikon Eclipse Ti microscope (Nikon Corporation, Chiyoda, Tokyo, Japan). A LED Hub (Omicron-laserage Laserprodukte GmbH, Rodgau, Germany) was used as a light source, and pictures were taken as 16-bit images with an ANDOR camera (Model No: DU-885K-CSO-#VP; Andor Technology, Belfast, UK) with a 60 X objective [60x/A/1.20 WI ∞/0.15–0.18 DIC N2 WD 1.0 objective (Plan APO VC Nikon CFI); Nikon Corporation]. Images were processed with VisiView software (Visitron Systems GmbH, Puchheim, Germany) and stored as TIF files. The mean background of respective experiments of individual channels was subtracted from all pictures in ImageJ [47]. A circular region of interest was created in ImageJ, which fit the rat nuclear size displayed as DAPI staining (Ø 11 µm). All nuclei of one region that collocated to neuronal MAP 2 staining were taken into consideration, and a mean value of average pixel intensity of pCREB staining was determined per region.