Chapter 2 72 Immunocytochemistry Cells were fixed for immunostaining using either 10 min fixation in 4% PFA (GAD65/67, CTIP2, MBP and OLIG2 staining) or by 10 minute fixation in ice cold 1:1 Acetone:Methanol. After fixation, cells were washed 6x 5 min with PBS and incubated in blocking buffer (PBS + 5% NGS + 0.3% Triton X-100 + 0.1% BSA) for 1 hour at RT. Primary antibodies were diluted in blocking buffer and incubated for 1 hour at RT followed by overnight incubation at 4˚C (see Table 2 for primary antibodies). The next day, cells were washed 6x 5min with PBS and incubated in secondary antibodies (Goat-anti-chicken/mouse/guinea pig/rabbit/rat Alexa Fluor 488/568/647) diluted 1:1000 in blocking buffer for 2 hours at RT. Cells were washed 6x 5 min with PBS, incubated in DAPI (1:1000 in PBS) for 1-2 min and embedded on microscope slides using Fluoromount G. Cells were imaged on a Leica DM6000B Fluorescent microscope, or on a Nikon Eclipse Ti confocal microscope for stainings of synapses and myelin. For GAD65/67 and CTIP2 stainings cells were scanned with a CellInsight CX7 HCA (ThermoFisher Scientific) and afterwards analyzed with Columbus software (Perkin Elmer). The proportion of GABAergic cells was determined by the percentage of MAP2+ neurons that were also GAD65/67+. As the neuronal differentiation gives rise to GABAergic and glutamatergic neurons(Nadadhur et al., 2017) we determined the proportion of glutamatergic neurons as the percentage of MAP2+ neurons that were not positive for GAD65/67. Immunohistochemistry Formalin-fixed paraffin-embedded tissue was sliced into 5 μm-thick sections. Immunostaining was started by rehydrating sections in xylene and alcohol. Endogenous peroxidase was quenched in 0.3% (w/v) H202 in PBS for 30 minutes, followed by heat induced antigen retrieval in citrate buffer (pH=6). Primary antibody GAD65/67 (1:1000, G-5163, Sigma) was incubated overnight at room temperature. The following day slides were rinsed and incubated with horseradish peroxidase labelled secondary antibodies for 1 hour and developed using 3,3′-diaminobenzidine (DAB, 1:50, DAKO) for 5 minutes. Sections were counterstained with haematoxylin, dehydrated with alcohol and xylene and mounted with Quick-D (Klinipath, 7280). Light microscopy pictures were taken with a Leica DM6000B microscope (Leica microsystems). Quantification of the number of GAD65/67 cells was done by manually counting the number of nuclei and the number of GAD65/67+ nuclei on 4 pictures (10x magnification) of the cortex.
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