DBH (Dopamine-beta-Hydroxylase) Antibody

Mouse hearts were perfused with PBS (pH 7.4) at RT, followed by cold fixative (4% paraformaldehyde + 0.2% picric acid in PBS). Tissues were fixed again for 2h at 4°C in cold 20% sucrose/PBS, and sectioned in a microtome. Slides was wrapped in aluminum foil and stored at −20°C. Slide-mounted heart tissue sections were washed 3 x 5 min in 0.1M PBS, permeabilized with 0.4% Triton X-100 plus 0.5% BSA for 20 min. Tissues were then incubated overnight with DBH antibody (1:1000 dilution), washed four times with 0.1M PBS (10 min each), permeabilized with 0.4% Triton X-100 + 0.5% BSA, and incubated for 1.5h with a secondary antibody. After washing, labeled tissue sections were viewed under a confocal microscope system. Labelled tissue sections showed clear labeling while the negative control sections (with our DBH antibody) showed only background fluorescence. This DBH antibody worked beautifully in our experiments!

We used 22806 in rat tissue.Brains were fixed using 4% paraformaldehyde, saturated in 30% sucrose and stored at -80. Brains were sectioned at 40 um using a freezing/sliding microtome and sections were stored in cryoprotectant at -20. The antibody clearly labeled fiber innervation in the cortical areas and innervation was easily measured via stereology. Brains were incubated overnight at RT at 1:500 in TBST +1% Goat Serum after blocking in 10% Serum in TBST. Vector biotinylated anti-rabbit was then used at a 1:500 dilution in TBST. After 1 hr ABC incubation, antibody was detected with DAB Nickel with a 10 minute development.

We used rabbit anti-bovine DBH (#22806) in an ICC Zenk protocol with sectioned zebra finch brain tissue and were very happy with the results. The antibody provided clear staining with minimal background. The antibody clearly labeled neuron bodies as well as tracks. Our protocol followed Sockman & Salvante 2007. Zebra finch brains were fixed using 4% paraformaldehyde, saturated in 30% sucrose and stored at -80. Brains were sectioned at 40 um using a cryostat and sections were stored in cryoprotectant at -20. Primary 1. 3x5 min wash in PBS 2. 15 min incubation in 0.1% sodium borohydride 3. 3x5 min wash in PBS 4. 30 min incubation in 0.5% H2O2 in PBS 5. 3x5 min was in 0.3% PBST 6. 1 hr incubation in 20% normal goat serum 7. 15 min incubation in Aviden solution 8. 3x5 min wash in 0.3% PBST 9. 15 min incubation in Biotin 10. 3x5 min wash in 0.3% PBST 11. 48 hr incubation at 4˚C at 1:16,000 in PBSTN Secondary 1. 1 hour incubation in secondary antibody at RT 2. Visualize using ABC kit and DAB 3. Dehydrate and coverslip