Veterinary session

To investigate the efficacy of proliferation markers anti Ki-67 and proliferating cellular nuclear antigen (PCNA) on equine conjunctival pseudotumor samples and their use in the calculation of proliferation index.

Following excision the tissue samples (n=13) they were fixed in 8% neutral buffered (in PBS, pH 7.0) formalin solution for 24 hours at room temperature, dehydrated in a series of ethanol and xylene and embedded in paraffin. Sections (3-4 μm) were cut from each normal, conventional tissue blocks and then deparaffinized in xylene and graded ethanol. The deparaffinized sections were treated with the primary antibodies Anti Ki-67, Anti Ki-67 clone BGX-297 and PCNA humanized antibodies for 60 minutes at room temperature after treatment with appropriate antigen retrieval process and microwave heat treatment. Immunohistochemical staining was performed using the streptavidin-peroxidase procedure. Antigen-bound primary antibody was detected using standard avidin-biotin immunoperoxidase complex. The chromogen substrate was 3,3' Diaminobenzidine (DAB) in each case. Mayer’s hemalaun was used for counter-staining. For each antibody a negative control with omission of the primary antibody was included. The 3-4μm thick sections were also routinely stained with hematoxylin.

Anti Ki-67 showed no affinity for the equine tissues, however both Anti Ki-67 clone [BGX-297] and PCNA humanized antibody used showed an affinity for equine tissue samples. HE staining showed an inflammatory cell population consisting of lymphocytes and plasma cells and marked reactive lymphoid hyperplasia which correlates with similar findings in the literature. The results show that PCNA appears to be a more sensitive than Ki-67 labeling in this case.

We can confirm that both the Anti Ki-67 clone [BGX 297] and proliferating cellular antigen (PCNA) can be successfully used on formalin fixed, paraffin embedded equine tissues , after antigen retrieval pretreatment for the estimation of proliferation index.