Staining and analysis was carried out as detailed elsewhere [13 (link)]. In brief, mid-sagittal cerebellar vibratome sections were blocked and permeabilized with 10% normal goat serum in PBS containing 0.2% Triton X-100 and immunostained with a mouse anti-calbindin D28k antibody (Sigma-Aldrich, 1:1000) followed by incubation with Alexa Fluor 555-labeled goat anti-mouse secondary antibody (Thermo Fisher Scientific, 1:1000).
Coronal forebrain sections were blocked and permeabilized as above, stained with rat anti-MBP (Merck Millipore, Burlington, VT, USA, 1:300), mouse anti-PV (Millipore, 1:1000), or mouse anti-GAD67 (Millipore, 1:200), and incubated with Alexa Fluor 555-labeled secondary antibody produced in goat (Thermo Fisher Scientific, all 1:1000). To visualize myelin, sections were incubated with FluoroMyelin Green Stain according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA, 1:300 dye dilution). Sections between Bregma 1.045 and −1.555 were employed for these analyses.
Pictures were taken with an Olympus AX70 microscope or Zeiss ApoTome2. For quantification of the Purkinje cell (PC) outgrowth, thickness of the molecular layer (ML) that reflects the dimension of the PC dendritic tree was determined at three different positions in lobules III, IV and V using ImageJ. PV positive neurons were counted in all layers of the somatosensory and retrosplenial cortex and normalized to the size of the analyzed area. For quantifying MBP, GAD67, and FluoroMyelin staining intensities, the respective integrated fluorescence signal intensities per area were measured using ImageJ software. Wt average values were set as 1.0. Blinding was achieved by attributing random numbers to the pictures. For each analysis, four brain sections per animal from 3–5 mice per experimental group were employed.
Coronal forebrain sections were blocked and permeabilized as above, stained with rat anti-MBP (Merck Millipore, Burlington, VT, USA, 1:300), mouse anti-PV (Millipore, 1:1000), or mouse anti-GAD67 (Millipore, 1:200), and incubated with Alexa Fluor 555-labeled secondary antibody produced in goat (Thermo Fisher Scientific, all 1:1000). To visualize myelin, sections were incubated with FluoroMyelin Green Stain according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA, 1:300 dye dilution). Sections between Bregma 1.045 and −1.555 were employed for these analyses.
Pictures were taken with an Olympus AX70 microscope or Zeiss ApoTome2. For quantification of the Purkinje cell (PC) outgrowth, thickness of the molecular layer (ML) that reflects the dimension of the PC dendritic tree was determined at three different positions in lobules III, IV and V using ImageJ. PV positive neurons were counted in all layers of the somatosensory and retrosplenial cortex and normalized to the size of the analyzed area. For quantifying MBP, GAD67, and FluoroMyelin staining intensities, the respective integrated fluorescence signal intensities per area were measured using ImageJ software. Wt average values were set as 1.0. Blinding was achieved by attributing random numbers to the pictures. For each analysis, four brain sections per animal from 3–5 mice per experimental group were employed.
Free full text: Click here
