Anti-Acid Sensitive Ion Channel 3 (ASIC3, DRASIC)

Applications: , Suitable for use in Immunohistochemistry and Immunocytochemistry. Other applications not tested. Recommended Dilution: Immunohistochemistry (perfusion fixed tissue): 1:50 Perfusion: 1) calcium-free Tyrode's solution, 2) paraformaldehyde-picric acid fixative, and 3) 10% sucrose in PBS as cryo-protectant. Desired tissues were dissected and stored overnight in 10% sucrose, PBS. , Immunocytochemistry: 1:50 ASIC3 transfected cells and primary neuronal cell cultures., Optimal dilutions to be determined by the researcher. Additional Information: Male Sprague-Dawley rats (b.wt. 100-150g) were anesthetized with sodium pentobarbital and perfused via the ascending aorta with: 1) 50ml of Ca2+-free Tyrode+s solution followed by 2) a formalin-picric acid fixative (4% paraformaldehyde with 0.4% picric acid in 0.16 M phosphate buffer, pH 6.9) and 3) 10% sucrose in PBS as a cryo-protectant. Tissues were rapidly dissected out and stored overnight in 0.1 M phosphate buffer , (pH 7.4) containing 10% sucrose. Slide-mounted tissue sections were incubated with blocking buffer for 1 hour at room temperature. Primary antibody was diluted in blocking buffer to the appropriate working dilution. Blocking buffer was removed and the slides were then incubated at 4°C for 18-24 hours with antibody. After rinsing in PBS 3 times sections were incubated for 60 minutes at room temperature with Cy3-conjugated secondary antibodies. After mounting in a mixture of PBS and glycerol (1:3) containing 0.1% p-phenylenediamine, sections were examined with a Nikon Microphot-SA epifluorescence microscope. Storage and Stability: May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.