Sample preparation: Tears were collected from the lateral canthus of mice using glass microcapillary tubes. One sample consisted of tears from both eyes of one mouse that were pooled in phosphate buffered saline (PBS) + 0.1% BSA and stored at −80°C until the assay was performed (Dogru et al., 2022 (link)). Hippocampus tissues of mice were isolated, snap-frozen in liquid nitrogen, and stored at − 80°C for protein extraction. The tissues rinsed with cold PBS to remove residual blood, weighed, and added with PBS containing protease inhibitor cocktail (1:9 w/v). Samples were then homogenized, centrifuged at 3500 ×g for 20 min, and supernatants were collected (Xue et al., 2021 (link); Dang et al., 2022 (link)).
The levels of inflammatory cytokines including tumor necrosis factor (TNF)-α, IL-1β, and IL-18 in hippocampi and tears were detected using Mouse TNF-α ELISA Kit (PT512, Beyotime), Mouse IL-1β ELISA Kit (PI301, Beyotime), and Mouse IL-18 ELISA Kit (PI553, Beyotime), respectively, as per company instructions.
The levels of inflammatory cytokines including tumor necrosis factor (TNF)-α, IL-1β, and IL-18 in hippocampi and tears were detected using Mouse TNF-α ELISA Kit (PT512, Beyotime), Mouse IL-1β ELISA Kit (PI301, Beyotime), and Mouse IL-18 ELISA Kit (PI553, Beyotime), respectively, as per company instructions.
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