Ab16030

Transfected neurons were treated and fixed as described above. After fixation, cells were rinsed in PBS and permeabilized with 0.25% Triton X-100 detergent (Bio-Rad Laboratories), followed by 2 rinses in PBS, and blocked with 5% BSA in PBS for 30minutes. Cells were rinsed with PBS again, and incubated with antibodies; (anti-vesicular glutamate transporter (VGLUT)1 (NeuroMab #75–066, 1:250), anti-SOCS3 (Abcam #ab16030, 1:500) or anti-pSTAT3 (Abcam #ab76315, 1:500) diluted in IHC-Tek (#IW-1001) for 2 hours at room temperature with mild shaking. Cells were rinsed with PBS three times and incubated in the appropriate Alexa Fluor secondary IgG antibodies (Thermo Fisher #A31571 and #A11035, 1:1000) for 2 hours at room temperature. Then, they were incubated in Hoechst (Invitrogen #3570, 1:1000) for 10 minutes. They were washed with PBS three times and mounted with Elvanol. Neurons for colocalization analysis were imaged as described in the Spine quantification section and juxtaposition of spines with presynaptic marker were quantified using ImageJ. For analysis of pSTAT3 and SOCS3 fluorescence intensity, in ImageJ, the z-stacks were summed, and background intensity were subtracted. Either whole neuron (Clover) or just nucleus (Hoechst) have been used as a mask and mean fluorescent intensity were reported due to the differences in neuron size.