David C. Rowlands

Microwave oven antigen retrieval applied to the immunostaining of cytopathology specimens.

Microwave oven antigen retrieval has been developed to extend the range of antibodies that can be used upon sections of fixed and processed tissue. It has the additional advantages of improving immunostain intensity and reducing background positivity. It can also be employed as an alternative to proteolytic digestion. In this study the effects of microwave oven heating upon immunochemical staining of cytopathological specimens with a range of selected antibodies have been investigated. Microwaving did not result in loss of cells, and there was no need to use adhesive‐coated slides. the method improved the staining intensity and reduced background with antibodies against a variety of antigens that are difficult or impossible to detect in formaldehyde‐fixed cytological material. Microwave heating was also used successfully as an alternative to trypsin digestion, and had the advantage of reduced morphological distortion. the technique was useful in demonstrating the soluble formalin‐sensitive antigen p19 on cytospins fixed in formaldehyde vapour. Microwave oven heating thus shows promise of extending the scope of immunostaining in clinical cytopathology.

IDENTIFICATION OF THE ANTIGEN RECOGNIZED BY THE MONOCLONAL ANTIBODY BU31 AS LAMINS A AND C

The murine monoclonal antibody BU31 binds to the nuclear membrane of many cell types. The expression of the BU31 antigen has previously been shown to have an inverse correlation with the proliferative index in lung tumours, defined by Ki67 staining. The distribution of BU31‐positive cells is now shown to parallel the distribution of non‐dividing cells in a range of normal human and rat tissues, although neuroendocrine cells and germ cells in the testis show no reactivity. Cells grown in culture and induced to undergo growth arrest show a higher level of labelling with BU31 than their proliferating counterparts. Confocal laser scanning microscopy reveals that the BU31 antigen is distributed predominantly along the nuclear lamina, with occasional internal foci. This distribution is very similar to that of the nuclear membrane proteins lamin A and lamin C, suggesting that the BU31 antigen and lamins A and C could be one and the same. Immunoblotting using recombinant lamin proteins confirmed this proposal. Moreover, a monoclonal antibody to the non‐proliferation‐associated antigen, statin, also recognizes lamins A and C. These data indicate that the demonstration of lamins A and C can be used to provide information on the proliferative activity of normal and neoplastic tissues. These data also suggest a role for nuclear lamins A and C during cellular quiescence, possibly through the reorganization and maintenance of nuclear structure, or more directly through interactions with the retinoblastoma gene product or related proteins.

Down-regulation but not phosphorylation of stathmin is associated with induction of HL60 cell growth arrest and differentiation by physiological agents

Stathmin is a cytosolic phosphoprotein that has an important but, as yet, undefined role in cell proliferation and differentiation. Induction of growth arrest and differentiation of HL60 cells to monocytes by phorbol 12‐myristate 13‐acetate is associated with rapid phosphorylation of the protein. Stathmin phosphorylation was not seen when HL60 cells were induced to differentiate to monocytes, by 1α,25‐dihydroxyvitamin D3, and to neutrophils, by all‐trans retinoic acid and granulocyte colony stimulating factor. In all the above instances, stathmin expression was down‐regulated. Thus, increased stathmin phosphorylation is not required for cell growth arrest or differentiation or down‐regulation of stathmin expression.

The effect of different fixatives and length of fixation time on subsequent AgNOR staining for frozen and paraffin-embedded tissue sections

SummaryThe AgNOR technique has been used extensively in studies investigating the possibility that the numbers and appearances of the intranuclear structures stained are markers of malignancy. The method has the advantage of being applicable to many different types of histological material, including paraffin-embedded tissue. However, it has been suggested that the visualization of AgNORs is dependent on the type and time of fixation employed. This study set out to measure this effect with the following commonly-used fixatives: acetone, absolute ethanol, methanol, Carnoys fluid, Bouins fluid, 4% glutaraldehyde, 10% neutral buffered formalin and 10% formol-saline. Both frozen sections and blocks of fresh tonsil were fixed for varying times, the blocks of tissue then being processed routinely. With the frozen sections AgNORs were easier to discern than in sections of paraffin-embedded tissue, and more intranucleolar AgNORs were visible when alcoholic fixatives were used than with aldehyde fixation. The effects of different fixatives on AgNOR appearance in paraffin sections is, however, more complex. Despite the variation caused by different fixatives, AgNORs could be demonstrated adequately with all the fixatives studied. It is concluded that fixation is not a limitation to the study of AgNORs provided that the time and type of fixative is controlled.

Silver staining of nucleolar organizer region associated proteins using polyethylene glycol as the protective colloidal developer

SummaryA simple modification to the silver staining technique for the demonstration of nucleolar organizer region associated proteins is described. Polyethylene glycol 20 000 is used instead of gelatin as the colloidal developer. This modified technique remains a one stage procedure that is quick and easy to perform. It results in reduced precipitate and less non-specific staining with specimens in which it had been previously difficult to demonstrate these intranuclear silver staining structures.