Biotinylated goat anti rabbit igg

Fly brains were immunostained with rabbit anti-5HT polyclonal antibody (1:200, S5545, Sigma-Aldrich) at 25°C for 24 h. After three washes in PBS-T, the samples were incubated in biotinylated goat anti-rabbit IgG (1:200, 31,822, Thermo Fisher Scientific) at 25°C for 1 day. The brain samples were then washed and incubated in Alexa Fluor 635 streptavidin (1:500, S32364, Thermo Fisher Scientific) at 25°C for 1 day. After three-time extensive washing, the brain samples were cleared and mounted in FocusClear (FC-101, CelExplorer). Brain images were obtained using a Zeiss LSM 700 confocal microscope under the same confocal settings for each brain sample, and the images were further analyzed using ImageJ. Single optical sections were used to calculate the average intensity values per voxel of the 5HT immunopositive signals in the soma and fibers of DPM neurons, and the signals were normalized to the GFP intensity.

For bright field microscopy, immunohistochemistry assays were performed on every eighth section from the brains to identify the microglial activation using microglia markers (Iba-1 and TSPO). All washing and incubation procedures were performed using 0.1 M PBS unless stated otherwise. The sections were washed three times and treated with 1% H2O2 before being incubated for 2 h in the blocking solution (2% goat serum) to block the non-specific antigen-binding sites. They were then incubated in the primary rabbit-anti-Iba1 antibody (1:500, Wako, # 019-19741) and rabbit anti TSPO (1:500, Merck, #ABC139) solutions for 2 days at 4°C and, subsequently, in the secondary antibody (1:200, biotinylated goat anti-rabbit IgG; Life Technologies, #656140) for 2 h. The sections were washed three times and ABC solution (1:250, Vector Laboratories, # PK-6100) was applied for 2 h. The sections were then incubated in the developer solution containing 0.4 mg/ml DAB and 0.0006% hydrogen peroxide until an optimal color developed. Then, the sections were washed, mounted, dehydrated, coverslipped, and their images were captured with a Zeiss microscope (MBF Bioscience).

For bright field microscopy, immunohistochemistry assays were performed on every 6th section of the brains to identify microglial activation using microglia markers (Iba-1 and TSPO). All washing and incubation procedures were performed using 0.1 M PBS unless stated otherwise. The sections were washed 3 times and treated with 1% H₂O₂ before being incubated for 2 hrs in the blocking solution (2% goat serum) to block non-specific antigen binding sites. Then they were incubated in the primary rabbit-anti-Iba1 antibody (1:500, Wako, # 019-19741) and rabbit-anti-TSPO (1:500, Merck, #ABC139) solutions for two days at 4 °C and subsequently in the secondary antibody (1:200, biotinylated goat anti-rabbit IgG; Life Technologies, #656140) for 2 hrs. The sections were washed 3 times and ABC solution (1:250, Vector Laboratories, # PK-6100) was applied for 2 hrs. Sections were then incubated in the developer solution containing 0.4 mg/ml DAB and 0.0006% hydrogen peroxide until an optimal color developed. In the end, the sections were washed, mounted, dehydrated, coverslipped and images were captured with a Zeiss microscope (MBF Bioscience).

Plates were washed 5 times with PBST, and 100 μL of a protein A purified rabbit IgG (10 μg/mL) against crude OV adult ES antigen extract was added to each well and incubated at 37°C for 2 hours. After washing three times, 1:4,000 diluted biotinylated-goat anti rabbit IgG (Invitrogen, CA, USA) in PBST was added and incubated at 37°C for 1 hour. Thereafter, the plates were washed 3 times with PBST, and 100 μL/well of streptavidin horseradish peroxidase (HRP) conjugate (Zymed, CA, USA) was added diluted 1:10,000 in PBST. After incubation for 30 min, the plates were washed 3X with PBST, and the substrate solution Orthophenylenediamine hydrochloride (Sigma, St. Louis, MO, USA) was added to wells and incubated for 20 min in the dark at room temperature. The reaction was stopped by the addition of 2M sulfuric acid (H₂SO₄), and the plates were read on an absorbance reader (Tecan, Austria) at the optical density (OD) at 492 nm.
Two well-trained laboratory personnel were responsible for execution of the sample analysis of both tested samples (index cases) and reference standard tests and they were analyzed simultaneously. During the sample execution, the sample IDs were blinded and the laboratory staffs had no knowledge of the sample subjects.