Introduction

Detection of proliferating cells In situ, has paramount importance in various experimental and clinical scenarios. Particularly in case of cancer, the evaluation of cellular proliferation is being used extensively to prognosticate the diseases (Webster et al., 2007). Immunohistochemical analysis of different proliferation-associated proteins has been instrumental to identify and quantify the proliferating cells in situ. The most commonly used protein markers to evaluate cellular proliferation include proliferating cell nuclear antigen (PCNA), Ki67 and argyrophilic nucleolar organizing region (Ag-NOR) (Mukaratirwa, 2005; Webster et al., 2007). Among them, Ki67 is one of the important and frequently used indicators of cell proliferation and a promising marker for human and animal cancer diagnosis and prognosis. It is a non histone nuclear protein and has two protein isoforms with molecular weights of 345 and 395 kDa (Li et al., 2015). This protein is present during all active phases of the cell cycle (G₁, S, G2 and M), but absent in resting cells (G₀) (Gerdes et al., 1983; Wang et al., 2014). The protein has a half-life of only 1‑1.5 h. In later phases of mitosis (during anaphase and telophase), a sharp decrease in Ki67 levels occurs (Modlin et al., 2008). The study was aimed for detecting the usefulness of two different clones of Ki67 antibodies (SP-6 and MIB-5) to detect dog Ki67 protein expression in different tissues. Through immunohistochemical analysis, we found SP-6 as a suitable antibody to detect dog Ki67 in a reliable manner.

Materials and Methods

Collection and Preservation Of Samples      

For conducting the present study we took two different tissue samples, first one was a normal uterine tissue as a positive control and second was a mammary tumour. Uterine tissues were collected from apparently healthy female dogs presented for sterilization to the Department of Surgery and Radiology, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar. Fresh mammary tumour tissues were obtained from two female dogs those were presented for surgical excision/ mastectomy. As samples were collected from surgically excised portions from the operated animals, it did not seek any animal ethical committee approval. However, before collecting the samples, we have taken the prior consent of the owners. The tissue samples were immediately fixed in 10% neutral buffered formalin, dehydrated through graded alcohol solutions, embedded in paraffin, cut into 4-μm-thick sections.

Immunohistochemical Study of Ki67

Deparaffinized sections were hydrated in a graded series of alcohol solutions for 5 min each (100%, 95%, 70% ethanol) followed by washing in distilled water. Sections were incubated in antigen retrieval (boiling the sections at 100°C in steam cooker for 20 min), treated with 3% H₂O₂ for 15 min to block endogenous peroxidase followed by two PBS wash. Blocking with normal horse serum was performed for 30 min in humid/moist chamber. The primary Monoclonal Ki67 antibody (clone SP6, VP-RM04, Vector laboratories and MIB-5 M7248, Dako) were applied on the slides and incubated in humid chamber 2 hr/ overnight in refrigerator at 4ºC followed by two washings in PBST for 5 min each. Secondary biotinylated antibody was then applied and kept for 45 min. Then slides were washed with PBST twice for 5 min each followed by application of ABC reagent for 30 min. The antigen-antibody-peroxidase reaction was developed with a freshly prepared 3, 3’-diaminobenzidine (DAB) solution. Sections were later washed in distilled water to stop DAB reaction. Counter staining was done with Mayer’s haematoxylin for 2 min and Scott’s tap water application followed by washing in distilled water for 5 min. Finally the slides were dehydrated in ascending grades of alcohol, cleared in xylene and mounted with aqueous mounting solution (Vecta Mount TMAQ; Vector laboratories, Inc.; Burlingam, CA, USA). For each slide, a negative control was run by replacing the primary antibody with PBS buffer. All slides were observed under Leica DM500 light microscope and representative photographs were taken. A qualitative estimation of Ki67 based on the staining intensity of positive cells was performed.

Result and Discussion

As discussed earlier, Ki67 antibody is one of the important markers used in invasive cancer evaluation that classically expressed as a nuclear protein in all phases of cell cycle, except G0 (Ciulla et al., 2009). In the present study the two different clones of Ki67 antibody (SP6 & MIB-5) revealed different pattern of immuno reactivity towards a similar tissue sample. In the canine normal uterine tissue section, some surface epithelial cells showed dark brown, homogenous or punctate staining, limited exclusively to the nucleus in case of Ki67 clone SP6 (Fig.1a and 1b).

Fig.1a & 1b: Section of normal canine uterus showing positive immuno-staining for Ki67 (SP6)  antigen in the nucleus of surface epithelial cells

Similar nuclear pattern of staining was obtained in case of canine mammary tumour sections that revealed many Ki67 positive cells with clone SP6 (Fig. 3a and 3b). However, staining with clone MIB-5 revealed strong cytoplasmic reactivity in both the tissue sections. In case of uterine tissue the cytoplasmic staining with MIB-5 was mostly confined to the glandular epithelium of the endometrial glands, not in the surface epithelium (Fig. 2a and 2b).

The cancer cells in the mammary tumour section showed faint cytoplasmic staining pattern (Fig. 4a & 4b).The nuclear staining obtained in both the tissues (uterus and mammary tumour tissue) with clone SP6 is not reported earlier.

Although SP6 is specific for human tissues, it is found to be valuable aid for canine oncology. In the present study, exclusive cytoplasmic immunostaining of Ki67 (clone MIB5) that was observed in the glandular epithelium of endometrial glands and cells of mammary tumour sections indicates its possible nonspecific reactivity against an unknown protein. Similar kind of nonspecific reactivity of monoclonal antibodies has been reported in several other articles (Martin et al., 2001; Ciulla et al., 2009; Faratian et al., 2009; Wang et al., 2014).

As discussed earlier, human and canine Ki67 protein sequence has only 50% similarity (Sysel et al., 2015). However, detection and quantification of canine Ki-67 using human-specific primary antibody has been widely used in literature. Most of the commercially available Ki67 antibodies are generated by using human Ki67 peptide of purified proteins. Only a few of the antibodies have been experimentally validated for their usefulness to detect canine Ki67 through IHC.  Nonspecific staining in IHC analysis is a serious concern. Hence, antibodies should be thoroughly validated to check their sensitivity and specificity. Keeping this point in view, we have experimentally provided the evidence about the cross reactivity of SP6 antibody against canine Ki67 protein. In future, this antibody might be reliably used for clinical and research purpose where dog is the subject of investigation.

Conclusion

The Ki 67 antibody (clone-SP6) has cross- reactivity against canine Ki 67 protein.  The cytoplasmic immunostaining obtained in case of MIB5 antibody suggested some nonspecific reactivity towards an unknown canine protein that can be investigated further.

References

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