Rabbit anti mouse hrp

To confirm the synaptic changes after enzymatic or genetic manipulation of PNNs and hibernation like state. Series of pre and postsynaptic markers were measured. To separate proteins the Tris-glycine 4–15% precast gel on BioRad miniprotean aperture was used. Transfer was done on PVD membrane. Applied antibodies against postsynaptic markers: PSD95 (1:1000, Abcam, Cambridge, UK), GAD65/67 (1:3000, Abcam). Applied antibodies against presynaptic markers: vGLUT1 (1:1000, SYSY), vGAT (1:1000, Synaptic Systems), SNAP25 (1:1000, Synaptic Systems). Secondary antibodies goat anti rabbit-HRP (1:15,000, Abcam), rabbit anti mouse –HRP (1:15,000, Abcam) and goat anti-guinea pig-HRP (1:5000, Abcam) were used to visualize proteins of interest.

To determine the expression of ICAM1, Vero cells were fixed by treatment with ice cold 50% MeOH/50% acetone for 20 min and after washing, cells were incubated for 1 h with an antibody directed to ICAM1 at a 1:250 dilution (Ab2213, Abcam, Amsterdam, The Netherlands). After washing, cells were incubated with rabbit anti-mouse HRP (1:1000 dilution, Abcam, Amsterdam, The Netherlands) for 1 h and subsequently washed again. As a read-out, AEC ready-to-use (Abcam, Amsterdam, The Netherlands) substrate was added for 10 min. As a negative control, non-transfected Vero cells were used.