Mice were killed by CO2 anesthetization followed by cervical dislocation. Brains and spinal cords were carefully dissected and post fixation for at least one week was carried out in 4% phosphate-buffered formalin at 4 °C before the tissue was embedded in paraffin. Immunohistochemistry was performed on 4 μm paraffin sections as described previously [38 (link)]. The following antibodies were used: anti-Aβ antibody 24311 (1:500, [41 (link)]), IBA1 (1:500; #234004, Synaptic Systems) and GPNMB (1:500, Santa Cruz). Biotinylated secondary anti-rabbit, anti-guinea pig and anti-goat antibodies (1:200) were purchased from Dako or Jackson Immunoresearch. The staining was visualized using the avidin-biotin complex method with a VECTASTAIN kit (Vector Laboratories) and diaminobenzidine (DAB) as a chromogen providing a reddish-brown color with hematoxylin as nuclear counterstain.
For double-immunofluorescence staining, polyclonal goat anti-GPNMB antibody (1:500, AF2330, R&D Systems) was combined with IC16 (1:1000, against the N-terminus of Aβ), GFAP (1:500, #173004, Synaptic Systems), NeuN (1:300, MAB377, Millipore) and IBA1 (1:500, #234004, Synaptic Systems), respectively. The staining was visualized using Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies (1:750, Jackson Immunoresearch) and analyzed using a Nikon Eclipse Ti-E fluorescent microscope.
For double-immunofluorescence staining, polyclonal goat anti-GPNMB antibody (1:500, AF2330, R&D Systems) was combined with IC16 (1:1000, against the N-terminus of Aβ), GFAP (1:500, #173004, Synaptic Systems), NeuN (1:300, MAB377, Millipore) and IBA1 (1:500, #234004, Synaptic Systems), respectively. The staining was visualized using Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies (1:750, Jackson Immunoresearch) and analyzed using a Nikon Eclipse Ti-E fluorescent microscope.
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