Bdnf elisa kit

Curcumin extract (90%) was purchased from Sisco Research Laboratories (SRL) Pvt. Ltd., (Mumbai, Maharashtra, India). High Capacity cDNA kit, phosphate buffer saline (PBS), formaldehyde, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), trichloroacetic acid, thiobarbituric acid (TBA), butanol, nitroblue tetrazolium (NBT), disodium hydrogen phosphate, sodium citrate, and hydrogen peroxide (H₂O₂) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Mouse brain-derived neurotrophic factor (BDNF) ELISA kit and mouse CREB ELISA kit were purchased from Ray Biotech (Georgia, GA, USA). Mouse Synapsin ELISA kit and mouse DCX ELISA kit were purchased from Gentaur (Kampenhout, Belgium). RNA Isolation Reagents and TRIzol were purchased from Sigma Aldrich (St. Louis, MO, USA). SYBR Green was purchased from Applied Biosystems, MA, USA. NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), Polytron tissue homogenizer (Thomas Scientific, Swedesboro, NJ, USA), ELISA reader (Trans Asia Pvt. Ltd., Mumbai, Maharashtra, India), and UV spectrometer (Perkin Elmer, Waltham, MA, USA) were used during the conduction of experimental procedures.

Eight weeks after PBS or A91 immunization, animals were euthanized with an overdose of sodium pentobarbital (80 mg/kg) and the SC samples were rapidly excised. Reagents, samples, and standards were prepared according to the instructions of rat IL‐4 ELISA Kit (Cell Applications), IL‐10 ELISA Kit (RayBiotech), BDNF ELISA Kit (Ray Biotech), GAP‐43 ELISA Kit (Cusabio), and TNF‐α ELISA Kit (OriGene). Briefly, 100 µL standard or 30 µg total protein samples were added to each well and incubated for 2 hours at 37°C. The liquid of each well was removed and not washed. Afterward, 100 μL of biotin antibody (1×) was added to each well and incubated for 1 hour at 37°C. Then, it was aspirated and washed three times. One hundred μl HRP avidin (1×) was added to each well and incubated for 1 hour at 37°C. Subsequently, it was aspirated and washed five times and 90 µL of TMB substrate was added to each well. Posteriorly, the samples were incubated and protected from light for 15‐30 minutes at 37°C. Finally, 50 μL Stop solution was added to each well and read at 450 nm within 5 minutes.

Two months after therapeutic intervention, animals were euthanized with an overdose of pentobarbital sodium (80 mg/kg) and then, SC samples were rapidly excised. Reagents, samples, and standards were prepared according to the instructions provided by the manufacturer: IL-4 ELISA Kit (Cell Applications, San Diego, CA, USA), IL-10 ELISA kit (RayBiotech, Norcross, GA, USA), BDNF ELISA Kit (Ray Biotech, Norcross, GA, USA), neuromodulin (GAP-43) ELISA kit (CUSABIO, Houston, TX, USA) and TNFα ELISA kit (Origene, Rockville, MD, USA). Briefly, 100 μl standard or 30 μg total protein sample were added to each well and incubated for 2 h at 37°C. The liquid of each well was removed and not washed. Afterwards, 100 μl of Biotin-antibody (1x) was added to each well and incubated for 1 h at 37°C, followed by aspiration and washing for 3 times. One hundred μl HRP-avidin (1x) were added to each well and incubated for 1 h at 37°C. Subsequently, it was aspirated and washed 5 times and, 90 μl of TMB substrate were added to each well, incubated and protected from light for 15–30 min at 37°C. Finally, 50 μl Stop solution were added to each well and read at 450 nm within 5 min.