Anti-CGRP antibody (ab36001)

Expression of CGRP and P2X₃ in rat dorsal root ganglia.

Immunohistochemical detection of CGRP (left panel, ab36001) and P2X₃ (middle panel) on cryosections of a dorsal root ganglion. The right panel shows the merged picture; the white arrows indicate neuronal co-expression of CGRP and P2X₃.

Animals were transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). The DRGs (Th13-L2 to L6-S1) were dissected out and post-fixed for 30 min. After PBS washing, DRGs were incubated in 0.01M PBS with 20% sucrose at 4°C overnight and embedded in OCT medium for cryopreservation. Cryosections (9 μm) were thaw-mounted on poly-L-lysine-coated microscope slides. As a blocking step, cryosections were incubated for 1 h with 0.01M PBS (pH 7.4) containing 0.05% thimerosal (PBS) supplemented with 10% normal horse serum (NHS) and 1% Triton X-100. Goat polyclonal anti-CGRP (calcitonin gene-related peptide; ab36001, 1/2500; Abcam) and rabbit polyclonal anti-P2X₃ (1/500) were diluted in PBS supplemented with 10% NHS and applied overnight at room temperature. After rinsing in PBS, tissues were incubated with Cy3-labeled donkey anti-goat (1/4000) and FITC-labeled donkey anti-rabbit (1/300) diluted in 0.01M PBS (pH 7.4) and again rinsed in PBS.

Excised mouse bladders were fixed in 4% (w/v) paraformaldehyde dissolved in 100 mM sodium cacodylate (pH 7.4) buffer for 2 h at room temperature. In some cases, tissue was fixed by 100% methanol (4°C) for 5 min. Fixed tissue was cut into small pieces with a razor blade, cryoprotected, frozen, sectioned (5 µm), and incubated with primary antibodies (1/100 –1/500 dilution) for 2 h at room temperature (CGRP labeled red, afferent nerves). After washing away unbound primary antibody, the sections were incubated with a mixture of Alexa^® 488-conjugated secondary antibody (diluted 1/100), rhodamine-phalloidin (1/50), and Topro-3 (1/1,000). The sections were washed with PBS, postfixed with 4% (wt/vol) paraformaldehyde, and mounted under coverslips with p-diaminobenzidine-containing mounting medium. All immunofluorescent localization data shown are representative images of staining performed on at least three individual bladders.

IHC-P image of CGRP staining on rat spinal cord sections using ab36001 (1/3000).

The sections were deparaffinized and subjected to heat mediated antigen retrieval. The sections were then blocked using 1% BSA at 21°C for 10 min. Incubated with primary antibody at 21°C for 2 hours.

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ab36001 staining mouse colon tissue sections by IHC-Fr.

Sections were PFA fixed and blocked in 5% serum for 30 minutes at 24°C prior to incubating with ab36001, diluted 1/200 in PBS + 0.2% Triton X-100, for 18 hours at 4°C. The secondary antibody was an Alexa Fluor^® 546 conjugated, diluted 1/200.

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