Immunohistochemistry of Brain Samples

Immunohistochemistry was performed as described previously^{2 (link)}. Briefly, animals were anaesthetised with pentobarbital and then perfused with with ice-cold PBS followed by 4% PFA in PBS. The brains were extracted and fixed in 4% PFA in PBS overnight at 4 °C. The following day, brains were washed with PBS and 70 µm thick coronal sections were cut using a vibratome (Campden Instruments, UK). Sections were blocked with 10% FBS in PBS-T (PBS + 0.1% Triton X-100) for 1 h at RT followed by incubation with primary antibodies overnight. Antibodies used were: PGAP2 (Abcam, 1:500), GPX3 (Abcam, 1:1000), MACF1 (Abcam, 1:1000), NeuN (Chemicon, 1:1000), GFAP (Abcam, 1:2000), HOMER (Synaptic Systems, 1:500). The secondary, Alexa-Fluor conjugated antibodies were chosen accordingly to wave length emission (Molecular Probes). Sections were mounted with Fluorsave medium (Calbiochem) and photographs were taken using a Zeiss LSM 5 Exciter confocal microscope.