Detection of the Ki‐67 antigen in fixed and wax‐embedded sections with the monoclonal antibody MIB1

Abstract

Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki-67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki-67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki-67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki-67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nuclear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo--ultraviolet irradiated human skin. MIB1 staining correlated with semi-conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki-67. Thus, MIB1 represents a new anti-Ki-67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material.

Prognostic values of proliferating cell nuclear antigen (PCNA) and Ki-67 for radiotherapy of oesophageal squamous cell carcinomas

“…The need for caution has been noted when measuring PCNA LI using antibody PC10 on formalin-fixed human tumours (Yu et al, 1992;Coltrera et al, 1993;McCormick et al, 1993a;Yu and Filipe, 1993) including: (1) at least two intracellular forms of PCNA exist -a replicon-associated form and a non-associated form that is present in almost all cycling cells (Bravo and Macdonald, 1987); (2) the PCNA staining was found to be highly influenced by the primary antibody dilution and the fixation time, and it lacks a clear plateau (Wolf and Dittrich, 1992;Coltrera et al, 1993;McCormick et al, 1993b); (3) the long halflife of PCNA (approximately 20 h), results in stain remaining in cells which have recently left the cell cycle (Bravo and Macdonald, 1987). Therefore, several investigators recommended that Ki-67 LI is a reliable tool for the measurement of proliferation activity (Yu et al, 1992;McCormick et al, 1993a;Yu and Filipe, 1993) and is suitable for multi-institute cooperative studies. In those situations, wide variations in fixation times, formaline composition and antibody dilution among different laboratories could result in significant inter-laboratory differences.…”

Immunohistochemical detection of Ki67 in breast cancer correlates with transcriptional regulation of genes related to apoptosis and cell death

“…2 Ki67 expression shows a good relationship with growth fraction in several model systems, 3,4 and does not appear to be expressed during DNA repair processes. Hence, it is regarded as a marker of cell proliferation, and in invasive breast cancer, has been used to stratify patients into good and poor prognostic categories.…”

Prognostic significance of Ki-67 antigen and p53 protein expression in pancreatic duct carcinoma: a study of the monoclonal antibodies MIB-1 and DO-7 in formalin-fixed paraffin-embedded tumour material

“…The more recently developed MIB antibodies exhibit a pattern of immunostaining in formalin-fixed paraffin-embedded material identical with that of Ki-67 antibodies in fresh or frozen material (McCormick et al, 1993) and correlate with other markers of proliferation (Weidner et al, 1994). Thus, expression of the Ki-67 antigen has been used in studies on archival tumour material and has been correlated with patient survival time (Railo et al, 1993;Pinder et al, 1995).…”

“…The proliferating rate has often been described as a proliferating cell index (PCI) (Pinder et al, 1995) or has been scored subjectively (Railo et al, 1993;Lam et al, 1996). The PCI may be calculated with or without the use of interactive image analysis systems (Pinder et al, 1995;Lam et al, 1996).The more recently developed MIB antibodies exhibit a pattern of immunostaining in formalin-fixed paraffin-embedded material identical with that of Ki-67 antibodies in fresh or frozen material (McCormick et al, 1993) and correlate with other markers of proliferation (Weidner et al, 1994). Thus, expression of the Ki-67 antigen has been used in studies on archival tumour material and has been correlated with patient survival time (Railo et al, 1993;Pinder et al, 1995).…”