Immunohistochemical Profiling of Pathological Markers

Immunohistochemical staining of formalin-fixed and paraffin-embedded tissue for CD3 (mouse monoclonal, 1:25, Dako, Glostrup, Denmark), CD20 (mouse monoclonal, 1:700, Dako, Glostrup, Denmark), CD45 (mouse monoclonal, 1:800, Dako, Glostrup, Denmark), GFAP (rabbit polyclonal, 1:4000, Dako, Glostrup, Denmark), CD68 (supernatant from KiM1P hybridoma cells, kindly provided by Prof. Klapper, Institute of Pathology, Kiel, 1:5000), and TSPO (rabbit monoclonal, 1:400, Abcam, Cambridge, United Kingdom) was performed using the streptavidin-biotin method on an automated staining system (LINK48, DAKO). For double immunofluorescence (see Fig. 2b), slices were incubated as described previously [26 (link), 27 (link)] with antibodies against TSPO (1:250, rabbit anti-TSPO, NBP1–95674, AB_11015478, Novus Biologicals, Cambridge, UK), Iba1 (1:250, rabbit anti α Iba1, 019–19,742, Wako Chemicals, Neuss, Germany), and GFAP (1:1000, chicken anti GFAP, ab4674). Alexa Fluor 488/555 (1:800; Life Technologies) and DSB-X™ Biotin Goat Anti-Chicken IgG (1:800; Life Technologies) were used as secondary antibodies. Double immunofluorescence for TSPO and Iba-1 was performed using a preconjugated TSPO antibody (1:100; anti-PBR antibody [EPR5384] (Alexa Fluor® 647) (ab199836)).