Las 4000 imaging analyzer

Hippocampal tissues were isolated from mice of each group (n = 10) and then lysed with RIPA buffer containing protein phosphatase inhibitor (Sigma-Aldrich). Total proteins were extracted and transferred onto a PVDF membrane, followed by blockage for 2 h, and incubation with primary antibodies [IL-17A antibody (ab9056), iNOS antibody (ab15323), NF-κB p65 antibody (ab207297), and COX-2 antibody (ab15191)(all 1:1,000; Abcam) and secondary antibodies. In this study, β-actin served as the internal reference protein. The ultra-sensitive chemiluminescent liquid-based FujiFilm LAS 4000 imaging analyzer (FujiFilm, Tokyo, Japan) was used for visualization, and Image J (NIH, Bethesda, MD) was used to analyze the relative intensities of individual bands.

Total protein extraction was performed using the Whole Cell and Tissue Protein Extraction Kit, as recommended by the reagent manufacturer. The Bicinchoninic Acid Protein Assay Kit (Thermo Scientific™, Waltham, United States) was used to measure protein concentrations. One day after the MWM test, the hippocampus was separated from the brain (n = 7), and the proteins were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred from the gel to a polyethylene difluoride membrane. After blocking the membrane with 5% skim milk powder for 2 h, the membrane was incubated overnight with primary antibodies [Postsynaptic density protein 95 (PSD95), synapsin-I, synaptophysin (SYP), growth associated protein-43 (GAP43), BDNF receptor tyrosine kinase B (TrkB), phosphorylated TrkB (p-TrkB), extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), cyclic AMP response element binding protein (CREB) and phosphorylated CREB (p-CREB) were purchased form Abcam, and dilution in 1:800 or 1:1000 for testing]. After cleaning, the membrane was incubated with a secondary antibody for 2 h. Finally, the ultra-sensitive chemiluminescent liquid-based FujiFilm LAS 4000 imaging analyzer (FujiFilm, Tokyo, Japan) was used for visualization, and Image J (NIH, Bethesda, MD) was used to analyze the relative intensities of individual bands.

Total protein extraction was performed using the Whole Cell and Tissue Protein Extraction Kit (Thermo Fisher Scientific, United States) according to the manufacturer’s protocol (n = 5). Protein concentrations were measured using the BCA Protein Assay Kit (A53225, Thermo Fisher Scientific). The proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% skim milk powder for 2 h, the membrane was incubated overnight with primary antibody [p-IP3R (ab111615), total-IP3R (ab264281), MFN1 (ab221661), MFN2 (ab260861), GRP75 (ab171089), p-PERK (ab192591), ATF4 (ab184909), CHOP (ab11419), cleaved caspase-3 (ab32042), cleaved caspase-9 (ab184786), Bax (ab32503) and Bcl-2 (ab32124) were purchased from Abcam and diluted to 1: 1,000 before use). In this study, β-actin (ab8226, 1: 2000) served as the internal reference protein. After washing, the membrane was incubated with secondary antibody for 2 h. The ultra-sensitive chemiluminescent liquid-based FujiFilm LAS 4000 imaging analyzer (FujiFilm, Tokyo, Japan) was used for visualization, and Image J (NIH, Bethesda, MD, United States) was used to analyze the relative intensities of individual bands.