Cell blotting: techniques for staining and microscopical examination of cells blotted on nitrocellulose paper.

Standard cytological and histological staining procedures were successfully applied on nitrocellulose (NC) paper for staining a spectrum of cell types that were blotted on it. The staining procedures included a Papanicolaou method, a hematoxylin and eosin method, and an immunoperoxidase technique. The cell types consisted of various suspension and monolayer cultures as well as cells freshly prepared from tumor and nontumor tissues. Suspended cells were manually blotted on NC paper; they were fixed in one of the fixatives, which included 80% 2-propanol, 10% neutral-buffered formalin, Carnoy's solution, Bouin's fluid, and B-5 fixative; they were then stained by one or more of the above methods. After staining, the NC paper was dehydrated in absolute 2-propanol; made transparent by soaking in xylene; mounted permanently in a mounting medium (Permount) on a glass slide; and then examined under a microscope. The protein-blotting capacity of NC paper (Schleicher & Schuell) was compared with those of the conventional cell blotting media (Millipore and Gelman) which contained cellulose acetate. Bovine serum albumin was dot-blotted on the NC and the cellulose acetate filters, and stained for protein by amido black. As in cytological/histological staining, the amido black-stained filters were dehydrated in absolute 2-propanol, made transparent in xylene, and mounted in Permount. Densitometric tracings indicated that the NC paper bound the highest amount of protein. The feasibility of cytological/histological staining techniques on NC paper combined with its high protein-blotting capacity allowed in situ staining and microscopical characterization of baby hamster kidney cells binding to fibronectin blotted on the NC paper. The possible significance of these techniques in current cell biological research was discussed.