Histamine Antibody

I have used this antibody (#22939) to label the brains of ~20 different species of insects. Brains are embedded in agarose and sectioned on a vibrating microtome at 50-70um. Note that the 4 hour carbodiimide fixation step (see below) is ABSOLUTELY necessary for the labeling to work. Pre-adsorbing the antibody with KLH does not affect labeling (see Dacks et al 2010, J. Comp. Neurol.). This antibody does not have great penetration for using a vibrating microtome, but 50um sections with 1% TX-100 for incubation with the primary is sufficient. DAY 1 Dissect brain in saline Fix brains in 4% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in phosphate buffered saline (PBS; pH 6.9) for 4hrs at 4°C Post-fix overnight in 4% paraformaldehyde at 4°C DAY 2 3X 15min PBS washes Embed brains in agarose and section at 50µm 4X 15min in PBS with .5% Triton X-100 (PBST) room temperature (RT) Block tissue for 1hr in 2% IgG free Bovine Serum Albumin (BSA) Incubate sections in 2% BSA in PBST (1%TX-100) with 50 mM sodium azide (PBSAT) with 1:500 Histamine (Immunostar # 22939) for 24hrs at RT DAY 3 4 X 15min PBST at RT Block tissue for 1hr (2% BSA) Spin down secondary in PBSAT with 2% BSA for 15min at 14,000RPM Incubate in block (PBSAT) with 1:1000 Cy3 Gt Rb 2° overnight at RT DAY 4 2x 30min wash in PBST 2x 30min wash in PBS 5 min in 60% glycerol Mount in 80% glycerol Only use 1% Triton X-100 for primary, otherwise always use .5%.