The assessment of cellular proliferation by immunohistochemistry: A review of currently available methods and their applications

Abstract

Immunohistochemical methods using antibodies to cell cycle-related antigens may be used as a means of assessing various aspects of proliferation in tissue, and have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Currently, the most widely available antibodies for this purpose are antibodies to bromodeoxyuridine (BrdU), Ki67 and antibodies to proliferating cell nuclear antigen (PCNA). BrdU is a thymidine analogue incorporated during the S phase of the cell cycle, which can be introduced by 'in vitro' incubation, and monoclonal antibodies are available to display its localization. Ki67 demonstrates a nuclear antigen expressed in all phases of the cell cycle, except G0 and early G1, but can only be applied to frozen tissue. PCNA is a nuclear antigen which is essential for DNA synthesis, two commercially available antibodies to PCNA work in paraffin-embedded tissue, but may have different staining characteristics under different conditions of fixation. The main advantages and disadvantages of these different techniques are discussed, together with their main applications to date.

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.…”

Ossification of the mouse metatarsal: Differentiation and proliferation in the presence/absence of a defined growth plate

“…Nuclear staining for this antigen identifies cells in the S-phase of the cell cycle (Yu et al, 1992). The above protocol was followed except antigen unmasking was instead facilitated by bathing in 95°C citric acid (pH 6.0) for 10 min, followed by digestion with 0.1 units/ml chondroitinase for 10 min at 37°C.…”

Electrophysiological Properties of Pluripotent Human and Mouse Embryonic Stem Cells

“…Specifically, we measured DNA synthesis as an index for replication by quantifying BrdU incorporation into genomic DNA during the S phase of the cell cycle, which is proportional to the rate of cell division [22]. Application of TEA + significantly inhibited the proliferation of mESCs in a dose-dependent manner (Fig.…”