Blocking serum

Cells were seeded on 8-well slides for immunohistochemical analysis. After 24 h, control, mon (4 µM), rap (50 nM), and mon + rap dose groups were formed and incubated for 72 h. The slides were washed with PBS and fixed with 4% paraformaldehyde. Then, they were treated with 3% H₂O₂/methanol for 10 min, washed with PBS, and blocked with blocking serum (Thermo Scientific, Waltham, MA, USA). They were next treated with primary antibodies at +4 °C for one night and washed with PBS. After that, they were washed with PBS and treated with Alexa fluor 555-goat anti-rabbit (1:200, cat. no. FNSA-0095). The samples were next covered with antifade mounting medium (Enzo Biochem Inc., Farmingdale, NY, USA) containing DAPI and left to dry overnight [36 (link)]. Examination of sections marked using the dual immunofluorescence method was carried out based on images taken with a digital camera (Olympus DP71 CCD color camera, 1.5 million pixels) using an Olympus BX–FLA Reflected Light Fluorescence Attachment adapted Olympus BX50 microscope and a 40× objective. Five randomly selected areas from each group were photographed at 200× magnification.

Cells were seeded on 8-well slides for immunohistochemical analysis. After 24 h, control, mon (4 µM), rap (50 nM), and mon + rap dose groups were formed and incubated for 72 h. The slides were washed with PBS and fixed with 4% paraformaldehyde. Then, they were treated with 3% H₂O₂/methanol for 10 min, washed with PBS, and blocked with blocking serum (Thermo Scientific, Waltham, MA, USA). They were next treated with primary antibodies at +4 °C for one night and washed with PBS. Following this, a specific secondary antibody was applied to the primary antibody. After washing with PBS, the secondary antibody was labeled with the streptavidin peroxidase enzyme complex. Proteins gained visibility with the DAB chromogen. The primary antibodies used and their dilutions were as follows: PI3K (1:2000, cat. no. BT-PHS00765, BTLab, Birmingham, UK), p-PI3K (1:2000, cat. no. BT-PHS00765, BTLab), AKT (1:2000, cat. no. BT-AP00334, BTLab), and p-AKT (1:2000, cat. no. BT-PHS00006, BTLab). In order to make the nuclei visible, they were counterstained with hematoxylin, washed in tap water until the dye ran, and covered with entellan after passing through increasing series of alcohol [36 (link)]. From each group, five different fields were photographed randomly at 200× magnification with light microscopy, and all SH-SY5Y cells showing AKT/p-AKT and PI3K/p-PI3K positivity were counted.