Cooled ccd camera

Immunocytochemical analysis was carried out for the detection of cell-specific markers as previously described [10 (link)]. Briefly, paraformaldehyde-fixed cells or cryostat sections were incubated in phosphate buffered saline, containing 5% normal goat serum or normal donkey serum and 0%, 0.2%, or 0.4% Triton-X100, followed by an overnight incubation in antibodies at 4°C. The list of antibodies used is given as supplementary data (Table S2). Cells were examined for epifluorescence following incubation in IgG conjugated to Cy3/FITC. Images were captured using a cooled CCD-camera (Princeton Instruments NJ, USA) and Openlab software (Improvision, MA, USA). In some cases, images were acquired using a Zeiss ApoTome Imager M2 microscope (Axiovert 200M) and captured by cooled CCD-camera (Zeiss). Axiovision 4.8 software was used for further image processing. Quantification of specific marker is presented as percentage of total DAPI positive cells. Five fields of four chambers of chamber slide or three coverslips were counted per experiment. Results were derived from three separate experiments.

EGFP-expressing retroviruses were injected into the lateral ventricle at E11.5, E12.5, E13.5 and E14.5, respectively. Two days later, brains were processed and serially sectioned using a vibratome. Consecutive sections covering the entire MGE/PoA were collected, immunostained and mounted on gelatin-coated slides. All EGFP-labeled RGPs in the MGE/PoA of each section were traced using Neurolucida (MBF Bioscience) on an upright microscope equipped with epifluoresence illumination and cooled CCD camera (Zeiss). All traced sections were then aligned to recover the entire RGPs. At least six brains were analyzed for each time point. For the quantitative analysis, at least three animals for each condition were examined.
For each postnatal brain, the number of interneurons in the somatosensory cortex was stereologically analyzed on both hemispheres from four consecutive coronal sections using Neurolucida and Stereo Investigator (MBF Bioscience). Cortical laminar position was determined by cell packing density revealed by DAPI staining. Data were presented as mean ± s.e.m. and statistical significance was determined using Student's two-tailed t-test.