S36939

Immunofluorescence staining was performed as described previously [29 (link)]. Briefly, the brain tissue was sampled as described above. The 15-25 μm brain sections were obtained, incubated with phosphate buffered saline (PBS) solution comprising 0.1% Triton X-100 for 30 min, and blocked with PBS solution comprising 5% goat serum (16210064, Gibco, NY, USA) for 30 min. Then, the sections were incubated overnight at 4°C with primary antibodies: IBA1 (ab5076, 1 : 500, Abcam, Cambrige, USA) and CD68 (ab6640, 1 : 200, Abcam, Cambrige, USA). After incubation, the sections were rinsed in PBS for 3 × 5 min washes and incubated for 1 h at 25°C with corresponding secondary antibodies: Alexa Fluor 488 antirabbit IgG (A11034, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 594 antigoat IgG (A11058, Invitrogen, Carlsbad, CA, USA). Lastly, the sections were dyed in DAPI solution (S36939; Invitrogen, Carlsbad, CA, USA) for 15 min at 25°C. The images of all sections were captured blindly using an A1 Si confocal microscope (Nikon, Japan).

Tissue was fixed in 10% formalin, paraffin embedded, and sectioned at 5μm thickness by a microtome at the University of Chicago Human Tissue Resources Center. Sections were baked for 60 minutes at 60°C, deparaffinized, and rehydrated in xylenes and alcohol. Heat induced epitope retrieval was performed with citric acid-EDTA buffer pH 6.2 as recommended by the manufacturer of the LAMA4 antibody. Sections were blocked in 10% donkey serum (Abcam ab7475) and incubated with primary antibody (anti-laminin alpha 4, Novus NBP2-42393, 1:300 dilution) overnight at 4°C. An Alexa Fluor-488 conjugated secondary antibody was added for 1 hour at room temperature (Abcam ab150113). Following incubation, sections were washed, stained with DAPI (Invitrogen S36939), and sealed. Images were taken using Fixed-DSU Confocal at the University of Chicago Integrated Light Microscopy Core and quantification of fluorescent signal was determined using ImageJ. All images were processed equally and in an unbiased manner to remove intracellular LAMA4 signal and background noise before signal quantification and analysis. Macro code for ImageJ analysis can be found in the Supplementary Materials file.