13 lens or 20 × 0.8 lens, in which case individual images were stitched together. Images of ß-galactosidase-immunostained sections for colocalization are single optical sections 0.8–0.9 μm thick, scanned selleck chemicals with an LSM510, Axiovert 200M confocal microscope, acquired with a 20 × 0.75 lens or 40 × /1.4 oil-immersion lens. The scans were acquired sequentially to ensure that there was no carry over of signal between the channels. The images were quantified
using the ImageJ cell count plugin; data are averages of at least three sections from three mice each and shown as mean ± SEM % cells. Mice were briefly anesthetized with isoflurane inhalation and decapitated. The brains were dissected, Cell Cycle inhibitor frozen in −40°C isopentane and stored at −80°C till used. Frozen brains were brought to −20°C; 14-μm-thick coronal cryostat sections collected on MMI membrane slides (Molecular Machines and Industries, Glattbrugg, Switzerland), dried for 1 h and stored at −80°C till used. For laser dissection, slides were stored on dry ice, one slide at a time was stained with 1 mL bis-benzimide (Hoechst 33258, Sanofi-Aventis;
10 μg/mL in 70% isopropanol) for 1 min, briefly rinsed in 70% isopropanol, dehydrated for 1 min in 100% isopropanol and air dried. The amount of material needed for simple immunoblotting Adenylyl cyclase using enhanced chemiluminescence detection can consist of 1000 cells or even fewer, depending on the abundance of the protein and the sensitivity of the primary antibody. Selected areas of the amygdala were cut using MMI CellCut laser microdissection system and captured on 0.5-mL caps of collecting tubes (MMI) for a final area of 200 000 μm2, sufficient for one or two determinations. Tissue areas were collected between Bregma −1.34 and −1.58
except for ITCs, which were collected over a larger range. Generally 10–14 cuts for the medial (mITCs) and lateral ITCs (lITCs), two cuts each for the lateral (no distinctions were made between the lateral and capsular subdivisions) and medial divisions of the CEA, and one cut each for LA and BA were sufficient. Care was taken that when multiple cuts were used they did not overlap on the cap. The tubes were put on dry ice and stored at −80 °C till used. Brief hematoxylin staining was also compatible with subsequent steps of immunoblotting. The slides could be recut at least a few times without noticeable deleterious effects. Immunoblotting was based on a previously described technique (Martinet et al., 2004), with some modifications.