Figure 9.

Immunohistochemistry of carbonic anhydrase VI distribution in maturation-stage ameloblasts of the rat incisor. CA6 localizes primarily to membrane invaginations along the distal surface of ruffle-ended ameloblasts (bottom panel). Strong reactions for CA6 are also evident in some papillary layer cells, especially in areas near blood vessels. The maxillary incisors of 100 g male rats were perfused via the vascular system for 20 min with 4% paraformaldehyde + 0.1% glutaraldehyde in 0.8 M sodium cacodylate buffer + 0.05% calcium chloride, pH 7.2. The jaws were decalcified for 3 wks in disodium EDTA, washed, then processed for embedding in paraffin. The sections (~5 µm) were treated for antigen retrieval in 10 mM sodium citrate for 15 min by being microwaved prior to immunolocalization with the anti-CA6 antibody, which was custom-made by Affinity BioReagents (Thermo Fisher Scientific, Inc., Rockford, IL, USA). The CA6 antibody is an affinity-purified chicken anti-rat IgY (egg yolk) antibody raised against the peptide CGGERQSPIDVKRREVHFSS, from near the rat CA6 N-terminus (aa 41–60). The CA6 antibody was incubated at 1:1000 dilution overnight at 4°C. The secondary rabbit anti-chicken antibody was incubated for 30 min on the section and then revealed with an immunoperoxidase kit (Vector Laboratories, Burlingame, CA, USA). The slide was counterstained with toluidine blue. Key: BV, blood vessels; CT, connective tissue; PL, papillary layer; RB, ruffled border; RE, ruffle-ended ameloblasts; SE, smooth-ended ameloblasts; transition zones between ruffled and smooth-ended ameloblasts, RE<SE and SE<RE.