Methionine Enkephalin Antibody

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Excellent Results and Reliability

We have used this antibody to stain Met-enkephalin positive processes throughout the adult mouse brain to study enkephalinergic innervation patterns. We confirmed specificity of the antibody with CRISPR-mediated knockdown in vivo. The antibody works well in formalin-fixed tissue imaged at 30-50 microns.

Immunohistochemistry: Animals were anesthetized using FatalPlus (Vortech) and transcardially perfused with 0.1 M phosphate-buffered saline (PBS), followed by 10% neutral buffered formalin solution (NBF, Sigma, HT501128). Brains were quickly removed and post-fixed in 10% NBF for 24 hours at 4 °C, and then cryo-protected in a 30% sucrose solution made in 0.1 M PBS until sinking to the bottom of the storage tube (~48 h). Brains were then frozen in Tissue Tek O.C.T. compound (Thermo Scientific), coronally sectioned on a cryostat (CM3050S, Leica Biosystems) at 30 μm and the sections were stored in 0.1 M PBS at 4 °C. Floating sections were permeabilized in a solution of 0.1 M PBS containing 0.3% Triton X-100 (PBS-T) for 30 min at room temperature and then blocked in a solution of 0.3% PBS-T and 5% normal donkey serum (NDS) for 2 hours before being incubated with primary antibody (1°Ab rabbit anti-met-enkephalin [1:1000, ImmunoStar, 20065]); prepared in a 0.3% PBS-T, 5% NDS solution for ~16-20 h at room temperature. Following washing three times for 10 min each in PBS-T, secondary antibody (2°Ab Alexa-Fluor 647 donkey anti-rabbit [1:500, Thermo Scientific, A31573]), prepared in a 0.3% PBS-T, 5% NDS solution were applied for ~2h at room temperature, after which the sections were washed again three times for 5 mins each in 0.1 M PBS, then again three times for 10 min in 0.1 M PBS, and then counterstained in a solution of 0.1 M PBS containing DAPI (1:5,000, Sigma, D9542). Fully stained sections were mounted onto Superfrost Plus microscope slides (Fisher Scientific) and allowed to dry and adhere to the slides before being coated with Fluoromount-G Mounting Medium (Invitrogen, 00-4958-02) and cover slipped.