Human, Mouse, Rat
(tested or 100% immunogen sequence identity)
Leu-Enkephalin antibody was raised against synthetic Leu5-enkephalin peptide (H-Try-Gly-Gly-Phe-Leu-OH) conjugated through the N-terminal tyrosine to BSA.
Leu5-enkephalin. Cross-reactivity: Met5-enkephalin 0.93%, beta-endorphin 0.01% and beta-Lipotropin 0.01% Species Reactivity: Human, rat and mouse.
Immunohistochemistry: frozen sections 1:500-1:1,000 using immunofluorescence and 1:1,000-1:5,000 for indirect avidin-biotin. See protocol below. Immunohistochemistry Protocol:Anesthetize animals and perfuse them transcardially as follows:a) Flush with cold (4°C) oxygenated Calcium free Tyrodesb) Perfuse with 4% formaldehyde in 0.16M phosphate buffer at pH 6.9c) Perfuse with 10% sucrose solution in 0.1M phosphate buffer (pH 7.2)Sections: Cut 5-30um sections using a cryostat and mount them on subbed histological slides. Immunofluorescence:1. Dilute LS-C23076 with 0.1M PBS, pH 7.4, 1% BSA, 0.1% Triton X-100. 2. Incubate sections for 24-48 hours at 4°C.3. Wash in PBS (three times for 10 minutes). 4. Incubate for 1 hours at RT with properly diluted donkey anti-rabbit secondary antibody conjugated to a fluorescent probe such as FITC, Rhodamine or Cy3.5. Wash in PBS (three times for 10 minutes) and mount with a PBS/glycerol solution, 0.1% phenylenediamine to reduce fading. If not stained with FITC, sections can be dehydrated in grading alcohols (50%, 75%, 80%, 96% and 100%), cleared with xylene and mounted with DPX (Fluka, Ronkonkoma, NY). Staining can be visualized by using both conventional and confocal microscopy.Indirect Avidin-Biotin Peroxidase: 1. Incubate sections with 0.3% H2O2 in PBS for 15 minutes at RT to block endogenous peroxidase. 2. Rinse sections with PBS three times for 10 minutes. 3. Dilute LS-C23076 with 0.1M PBS, pH 7.4, 1% BSA, 0.1% Triton X-100. 4. Incubate sections overnight at 4°C and then wash in PBS (three times for 10 minutes). 5. Incubate sections with properly diluted biotinylated goat anti-rabbit secondary antibody diluted in PBS (do not add sodium azide!) for one hour at RT. 6. Rinse sections three times for 15 minutes and incubate sections with horseradish peroxidase-streptavidin complex properly diluted in PBS/0.1% Triton X-100 for 40 minutes at RT. 7. Wash sections with PBS and incubate them in substrate solutions (e.g. DAB, AEC, etc.) to achieve necessary intensity of ANP staining.