4 6 diamidino 2 phenylindole mounting solution

To perform the immunofluorescence assay, the treated wells were washed with PBS and fixed at RT for 30 minutes in 4% paraformaldehyde. After blocking with 3% normal goat serum and 1% bovine serum albumin (Vector Laboratory, Burlingame, CA, USA) for 1 hour, the well plate was incubated with primary antibodies to BDNF (1:500; Santa Cruz Biotechnology) and VEGF (1:500, Santa Cruz Biotechnology) at 4°C overnight. After plate washes, the plates were incubated with secondary Alexa Fluor 488-goat anti-rabbit IgG (1:400; Vector Laboratory) and Alexa Fluor 594-goat anti-mouse IgG (1:400; Vector Laboratory) antibodies for 1.5 hours at RT. Cells were counterstained with a 4′,6-diamidino-2-phenylindole mounting solution (Vector Laboratory), and slide images were analyzed by fluorescence microscopy.

Briefly, the cells were seeded on 15 mm microscope cover glass (Paul Marienfeld GmbH & Co., KG, Lauda-Königshofen, Germany) at a density of 5×10⁶ cells/well in 12-well plates and grown overnight, followed by pre-treatment with essential oil and LPS stimulation for 24 h. For antibody labeling, the cells were first fixed with 37% formaldehyde and 95% ethanol (1:4) for 15 min at room temperature, followed by washing with 1X PBS three times (5 min/wash) and blocking with 1% BSA (Bioshop, Canada, Inc., Burlington, ON, Canada)/1X PBS for 1 h at room temperature. The cells were probed overnight with a 1:100 ratio of diluted antibody (p-p65) at 4°C. Following washing with 1X PBS four times (7-10 min/wash), the cells were blocked with 1:250 diluted anti-rabbit Alexa fluor 594 conjugate Red at room temperature for 1 h. The cells were then washed with 1X PBS and mounted with 4′,6-diamidino-2-phenylindole mounting solution on slides purchased from Vector Laboratories, Inc. (Burlingame, CA, USA) and were designated for confocal image analysis. All confocal images were captured with a ×20 oil objective (numerical aperture 3.5) lenses of Olympus Fluoview FV1000. FV10-ASW 3.1 viewer software (Olympus Corporation, Tokyo, Japan) was used to extract the images.