Sections were thoroughly washed in tris-buffered saline (TBS), then incubated in 10% methanol, 2% hydrogen peroxide solution in TBS for 30 min followed by washing and incubation in 4% normal donkey serum and 0.2% Triton X-100 in TBS. Sections were incubated in an earlier characterized rabbit-anti DAGL∝ at 1:2000 dilution overnight at room temperature (Katona et al, 2006). Sections were washed and then incubated in biotin-conjugated donkeyanti rabbit at 1:500 for 2 h, followed by HPR-conjugated streptavidin at 1:16000 for 90 min (Jackson ImmunoResearch, West Grove, PA). Sections were developed using diaminobenzadine substrate with nickel/cobalt heavy metal intensification as described earlier. For immunofluorescence, rabbit-anti DAGL∝ was used at 1:500, followed by 2 h incubation in Cy-3-conjugated donkey-anti rabbit at 1:250 for 2 h (Jackson ImmunoResearch).
