A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Abstract

Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tissues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.

Rate of Apoptosis in Feline Mammary Tumors is not Predictive of Postsurgical Survival

“…9 The ability of thiazines to be employed as Schiff-type reagents for the staining of DNA and carbohydrates has been previously described. 4,9,34 Quantitative evaluation of mitotic activity using an image analyzer is a rapid method of expressing mitoses per cell number (i.e., mitotic index), if combined with a stain such as toluidine blue, which has the advantage of staining neither the cytoplasm nor the stroma but only the nuclei. 3 Apoptosis determination in neoplasms is usually accomplished by 2 methods: morphologic detection of apoptotic bodies by light microscopy and in situ 3Јend labelling.…”

Prognostic Value of Histologic Stage and Proliferative Activity in Canine Malignant Mammary Tumors

“…b Toluidine blue stain A previously described modification was used. 8 The sections were dewaxed in toluene, rehydrated, and then hydrolyzed in 5 N HCl for 20 min at RT, rinsed twice (5 min each) in DW, and immersed for 10 min in a solution of 0.5 g toluidine blue O in 100 ml of bleach solution (90 ml of double-distilled water, 5 ml 1 N HCl, 5 ml 10% sodium metabisulphite). The sections were then immersed twice (1 min each) in bleach solution, rinsed in DW, dehydrated, and mounted with dibutylphthalate-xylene.…”

The Feulgen reaction 75 years on

“…All the dyes considered as reliable Schiff-like reagents contained at least one free amino group reactive with SO 2 and tested positive for the Feulgen reaction. The fluorescent acriflavine (Böhm and Sprenger 1968;Takamatsu et al 1980;Mikel and Becker 1991) and the blue-shaded thiazines, thionin (Böcking et al 1995), and toluidine blue (Chieco et al 1993), are nowadays the most common substitute for pararosaniline in Schiff-type reagents for quantitative work or simple nuclear counterstaining. Notably, although these reagents have been found specific for apurinic DNA, their reaction mechanism and the structure of reaction products are not yet defined and may significantly differ from that of the original Schiff reagent (Gil and Jotz 1976).…”