Fluoro jade c working solution

FJC staining of MIF WT and KO mouse sections (30 μm) post TBI was performed as previously described [30 (link),31 (link)]. Briefly, brain sections from each mouse were organized into a series of 24-32 sections separated successively by 90 μm. Each series is representative of all parts of mouse brain except cerebellum. A series of 24-32 brain sections from each mouse were mounted onto Superfrost Plus microscope slides (Fisher Scientific, 12-550-15). After dry, slides were immersed in 0.01% NaOH prepared in 80% ethanol solution for 5 min following by 70% alcohol for 2 min and distilled water for 2 min. Slides were then transferred to 0.06% potassium permanganate and shaked for 10 min in the dark. After rinsing with distilled water, slides were incubated in 0.0001% Fluoro-Jade C working solution (Sigma, AG325) with 0.1% acetic acid for 10 min in the dark. After staining, the sections were rinsed with distilled water 3 times for 1 min each. After dry, slides were immersed in 50%, 70% and 100% ethanol for 2 min each and then cleared in xylene. Lastly, slides were mounted with DPX (Sigma, 317616) for image analysis. All brain sections were checked under the microscope and 5-10 representative images were taken from the cortex of each single mouse and neurodegeneration was quantified as FJC+ cell number/image field from 15-30 images of WT and KO mouse following TBI.