Some practical considerations in quantitative absorbance microspectrophotometry. Preparation techniques in DNA cytophotometry.

Abstract

An experimental review of the Feulgen and gallocyanine-chrome-alum stains for quantitative cytophotometry of DNA in tissue sections yielded information on the preparation and staining of tissue for quantitative absorbance microspectrophotometry. (1) Tissues routinely fixed in formalin are suitable for either stain. Specimens fixed with glutaraldehyde-containing fixatives are not satisfactory for Feulgen staining, nor are ethanol-fixed specimens, unless they are post-fixed in formalin. (2) The pararosaniline dyes, used in the Feulgen stain, are sufficiently pure to use if a solution of the dye in ethanol shows an absorbance peak at 543 to 546 nm. (3) The Feulgen stain provides good reproducibility when the staining solution is adjusted to pH 1.5. (4) Gallocyanine is the best stain to use on Bouin-fixed or glutaraldehyde-fixed tissues. (5) Where fixation is an option, Carnoy and methanol-formalin-glacial acetic acid are excellent fixatives that can be followed by either stain. (6) Selection of the thickness of a tissue section involves a compromise. Requirements of minimum nuclear overlap and sharp focusing favor a section thickness of 4 micron to 6 micron. On the other hand, the requirement for full nuclear thickness, as judged by absorbance equivalent to that of a touch preparation, demands sections as thick as 8 micron in the case of mouse liver. Within this range, the optimum thickness, therefore, is determined by the particular tissue, the range of its nuclear sizes and its packing density. (7) The refractive index of the mounting medium should be closely matched to that of the background structure of the tissue sections. For animal tissues, we found that media with refractive indices of 1.54 to 1.56 are suitable.