Characterizing Immune Cells in Multiple Sclerosis Brains

Human brain sections from two different MS patients (female RRMS 49 y.o. and female 33 y.o. RRMS) were stained following established protocols (12 (link), 30 (link), 31 (link)). After fixation with acetone and ethanol 70% at -20°C and blocking with 10% mixed donkey/goat serum, sections were incubated for 1 h at 20°C with primary antibodies diluted in 3% donkey serum: mouse anti-human granzyme-B (1/20, R&D Systems, Cat#MAB2906) and rabbit anti-human NogoA (1/100, EndMillipore, Cat# AB5664P). Adequate secondary antibodies for these antibodies were used: donkey rabbit RRX (1:500, Jackson ImmunoResearch, Cat#711-295-152) and goat anti-mouse AlexaFluor635 (1:250, Invitrogen, Cat#A31574). Then, sections were blocked again in 10% rabbit serum and stained using mouse anti-human CD4-FITC antibody (1:50, BD Biosciences, Cat#555346), followed by a secondary antibody rabbit anti-FITC (1:50, Invitrogen, Cat#A11090). Finally, nuclei were stained using DAPI. Sections were mounted on slides with Prolong Gold Antifade.