Probe Design and Labeling

Primer table

Combined ISH Immunofluorescent Immunohistochemistry

To avoid possible cross reactivity, each probe was designed to include regions of low nucleotide identity with other related family members, or other sequences located in the NCBI nucleotide database. The highest level of identity found for any probe was calculated to be 73% (Table II). To further ensure the probes had no cross reactivity with their closely related subfamily members, we also performed a dot-blot hybridization for each probe against the cDNA’s of each EAG family member. As predicted from similarity calculations, each probe proved to be highly specific for its intended EAG subunit when hybridized at the same stringency conditions used in the ISH protocol.

Combined In situ hybridization-immunoflourescence histochemistry.

The non-radioactive ISH protocol used was based on a modified radioactive ISH method developed by Dr. Harriet Baker (Weiser et al. 1994, Saganich et al., 1999). Briefly, 6-8 week old male rats were perfused intracardially with 100 ml of cold saline solution (0.9% NaCl with 0.5% NaNO2 and 1000u. Heparin), followed by 300 ml of cold 4% paraformaldehyde solution in 0.1M Phosphate buffer, pH 7.4. The brains were carefully removed, cut in blocks and post-fixed for 1 hour. Following post-fixing, the brains were washed several times in cold, 0.1M phosphate buffer (pH 7.4) and placed in 30% sucrose overnight. Slices were obtained on a freezing-microtome at 40 *m thickness and floating sections prehybridized at 60oC in a solution containing 60% formamide, 3.5X SSC, 5% Dextran Sulfate, 3.5X Denhardt's, 0.5 mg/ml denatured salmon sperm DNA, 0.2 mg/ml t-RNA, and 0.25 mg/ml SDS. After 1 hour of pre-hybridization, 50ng/ml of DIG-labeled RNA probe was added and the hybridization reaction allowed to proceed for 17 hours.

After hybridization, the sections were washed in decreasing concentrations of SSC (2X to 0.1X) buffer at 65oC followed by a single wash in buffer B1 (150mM NaCl, 100mM Tris, pH 7.4) at room temperature (RT). Sections were then treated for 1 hour at RT in buffer B1 + 10% normal sheep serum followed by over-night incubation at 4 oC with anti-DIG Fab fragments conjugated with alkaline phosphatase (AP) in buffer B1+1% normal sheep serum. When co-labeling for neuronal nuclear protein (NeuN), parvalbumin (PV), Glutamate Decarboxylase (GAD67), and/or Calbindin (Cb) was desired, the antibodies were added with the anti-DIG antibodies. Antibodies were used at the following concentrations: Anti-DIG Fab, 1:3000 (Roche); NeuN, 1:500 (Chemicon MAB377); PV, 1:500 (Sigma); GAD67, 1:2500 (Chemicon AB108); Cb, 1:500 (Sigma). Overnight incubation with antibodies were followed by 3x15 min. washes in buffer B1 followed by 2 hour incubation at RT with secondary antibodies (anti-rabbit Cy2 and/or anti-mouse Cy3 (Molecular Probes) in buffer B1 + 1% Normal goat serum + 0.1% BSA + 0.02% cold waterfish gelatin. Following treatment with secondary Ab's, sections were washed 3x15 min. at RT in buffer B1 followed by a single wash in DIG detection buffer (100mM NaCl, 100mM Tris, 50mM MgCl2; pH 9.5). DIG detection was carried out using the AP substrate NBT/BCIP (Roche) for 8-24 hours in DIG detection buffer. The reaction was stopped by rinsing sections 4x15min. in ddH2O and these mounted in 0.1X SSC, partially dried, and coverslipped in 50% glycerol on glass slides. Images were acquired using an Olympus Provis microscope equipped with a MagniFire digital camera. Fluorescent images were acquired using filter sets for Cy2 and Cy3.