TI - Results Proliferation in the incudomalleal joint . AB - Very few PCNA positive cells have been observed in the immediate layer of developing joints in the limb [20,42-44] . The joint region is thus thought of as a region of low proliferation . To see whether this situation is mimicked in the middle ear we looked at the distribution of proliferating cells during early formation of the incudomalleal joint . At E14.5 and E15.5 the malleus and incus are seen as separate structures with a clear joint in between . Using PCNA , the level of proliferation was high in the head of the malleus and incus but the joint region was negative ( Fig 1A , B , D , E ) . PCNA has a long half-life leading to its continuous expression in cells that are not actively dividing [45] . We , therefore , supplemented our results by using histone H3 immunohistochemistry . The H3 antibody detects PHOSphorylation of histone H3 , which is high during chromosome condensation as the cell enters the M-phase and has a short half-life . As expected , only a few cells were observed as positive in the middle ear using this method compared to using PCNA . The positive cells were concentrated in and around the ossicles but again excluded from the developing joint ( Fig 1C , F ) . Labelled cells were also located around the circumference of the eye as previously published using this method , indicating that the assay was working [38] . At E13.5 the malleus and incus are still continuous [1] . At this early stage using PCNA , proliferation was observed in the malleus and incus but the forming joint region was negative , reflecting the situation observed at later stages ( Fig 1G , H ) . Using the H3 antibody at this stage , the incus showed positive proliferating cells but the presumptive joint region appeared negative ( Fig 1I ) . The malleus also appeared negative , using the antibody to His3 , perhaps indicating a time delay between proliferation of the two ossicles .