Sections processed for immunoperoxidase staining were visualized and photographed with an Axioscope 2 microscope (Carl Zeiss AG) equipped with a color digital camera (AxioCam MRc5) and its corresponding software, AxioVision 4.5 (Carl Zeiss AG). Images of the whole hippocampus were taken with bright-field illumination using a 5× objective (NA 0.15).

Images of cleaved caspase-3 and DAPI samples were imaged with a Zeiss Axio Imager 2 fluorescent microscope (Carl Zeiss AG). Image stacks (five slices, 2-μm intervals) from CA3 and CA1 areas were taken with a 10× objective, with a scan zoom of 1× and image size of 1024 × 1024 pixels.

Immunofluorescence GABA and NeuN-stained tissue sections were imaged using a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss AG) with a 40× oil immersion objective with a numerical aperture (NA) of 1.4. Images were taken as z-stacks (five slices, 1-μm intervals) with a scan zoom of 1× for CA3 area and 0.5× for the CA1 area and an image size of 1024 × 1024 pixels.

For GABAergic synaptic cluster analysis, vesicular GABA transporter (VGAT), gephyrin and GABAARγ2 subunits were stained, and sections were imaged using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss AG). For glutamatergic inputs, vesicular glutamate transporter (vglut)1-2 and PV cells stained sections were imaged. A 63× oil immersed objective with a NA of 1.4 was used for synapse analysis. Images were taken as z-stacks (five slices, 0.2-μm intervals) with a scan zoom of 1.5× and an image size of 1024 × 1024 pixels. Imaging parameters were kept constant over all conditions. After acquisition, images were processed using 2D super resolution Airy Scan processing run in automated mode. image acquisition was conducted in the stratum pyramidale and stratum radiatum of the CA1 and CA3 region.

For analysis of PNNs around PV+ interneurons, double-stained sections were imaged using a Zeiss LSM 800 confocal laser scanning microscope (Carl Zeiss AG) with a 10× objective (NA 0.45) and taking image stacks composed of 22 slices at a z-intervals of 0.4 μm. Image acquisition was performed in the CA1 and the CA3 area of the hippocampus and image settings were kept constant between genotypes within every age.

Samples were imaged with a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss AG). Image stacks (six optical sections, 0.5-μm step size) from CA1 and CA3 stratum pyramidale and radiatum were acquired with at 40× objective, N.A. 1.4.

Unbiased counting of PV+, SST+, and NPY+ interneurons in the hippocampus was performed using the 10× objective (NA 0.45) of an Axioplan 2 bright-field microscope (Carl Zeiss AG) equipped with a digital camera (MicroFIRE, Optronics AG). First, the hippocampal CA1 and CA3 areas containing the different subregions: stratum oriens, stratum pyramidale, stratum radiatum and stratum lacunosum moleculare, were delineated with Mercator Pro software (Explora Nova) according to the mouse brain atlas (Paxinos, 2007). Subsequently, immunoreactive cells were counted in each of these areas with no distinction of subregions. Data collection was done in serial sections throughout the whole hippocampus, analyzed with a serial sampling fraction (ssf) of three for P7, four for P11, five for P14, and six for P21 and P60. Six animals per genotype and age were analyzed, except for SST analysis where only four animals per genotype were analyzed.

The total volumes (Vtot) of the CA1 and CA3 were calculated from the ssf, the areas delineated in every section (Ai – An; n = number of sections analyzed) and the section thickness (h) as follows:

Subsequently, the total number of immuno-positive cells (Qtot) in the CA1 and CA3 were calculated using the ssf and the positive cell numbers per section (Qi):

Total number of NeuN+, and GABA+ cells were quantified with the optical Fractionator using the Stereo Investigator software (v10.50, MBF Bioscience) equipped for fluorescence imaging, with a 63× lens (Zeiss 1.4 Oil). Neu+ and GABA+ cells were counted independently in a frame of 40 × 40 μm with a step size of 120 μm. Total cell numbers (N) were calculated using the formula: N=Q.(1asf)(1ssf), where Q is the total number of counted cells, and asf the area sampling factor. A mean of 200 cells was counted per animal. For more details on stereological estimates (Slomianka and West, 2005).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.