Increase in Sensitivity of the Rhodamine B Method for Keratinization by the Use of Fluorescent Light

Abstract

Rhodamine B was first used as a stain for cornification b y Adortinofti 1924, who presented two methods for eleidine and keratogenc substances i n which the dye was used together with either monophenylrosanilin o r Victoria blue. I n 1968, Liisberg published a method for demonstrating-cornified tissue which mas claimcd to he extremcly specific. Rhodamine H was uscd a t pH 3,6 after blocking of basophilic suhstances by toluidine blue.The present authors tried Liisberg's method on formalin fixed, paraffin embedded sections of oral mucosa and Iwratocysts, b u t found that they stained rather faintly and not absolntcly specifically. -4s rhodamine B has becn used as a fluorchrome for many years (Harms 1965), it w a s decided t o t r y to enhance the sensitivity of the method hy means of fluorescence microscopy.Formalin fixed, paraffin embedded sections of biopsies from oral lcucoplakias, Iteratocysts, oral lichen planus and oral carcinomas, containing lteratinizcd layers as evidenced i n haemalum-eosin stainings w c r e used as test ohjects. I n addition scrapings from the palatal and buccal mucosa were staincd after fixation i n 96 per cent ethyl alcohol.T h e rhodaminc H mas Gurr's "Michrome no. 407", and t h e toluidine hlue was "Toluidinblau nach Hoyer". Sections were mounted i n Eultitt. Fluorcsccnce microscopical examination was made with a Leitz Orthoplan microscope fitted with a n Osram HHO 200 W lamp. A 5 m m BG 12 filter was nscd for cxcitation and a Leitz K530 filter as a harrier.In all sections examincd the sensitivity of thc rhodaminc B method was significantly enhanced by fluorcsccnt blue light. The kcratinized laycrs showed an intense: brilliant yellow, secondary fluorescence ( Fig. 1 A + B). A distinct fluorescence was seen cven i n areas whcre the Iteratinizcd layer was very thin and the staining very difficult to evaluate in white light ( Fig. 2 A + B). Unstained sites showcd a greenish fluorescence, a sharp contrast between lteratinizcd and unlicratinized sites therefore being readily obtained. Unfortunately, striatcd muscle tissue, collagen fibres, and erythrocytes arc often partly stained hy rhodamine 13, the reaction being seen clearly b y white light. These limitations of t h e method do not, however, influencc the interpretations of the kcratinization of the epithelium.Leucoplakias with orthokeratinized epithelium showed an intcnse rhodaminc Epositive reaction confined to the lteratinized layers, with a distinct border adjacent to the granular layer. The reaction was more sharply demarcatemd and more uniform i n fluorescent light t h a n in white light. Some of thc prickle cells showed a weak cytoplasmic reaction i n fluorescent light which was hardly recognizable i n white light.Leucoplakias having a paralteratinizcd epithelium and a stratum granulosnm exhibited, in visible light a n irregularly rhodamine B-positive kcratinized laycr with negative pylinotic nuclei, and i n fluorcsccnt light, a regularly positive sharply demarcated keratinized laycr with negative nuclei.Incomplete paralteratosi...

Keratocystic Odontogenic Tumor of the Maxilla: Report of a Rare Case and Review of Literature

“…Strong reaction in parakeratinized KCOTs suggested an alteration in gene expression that is also observed in BCCs but not in normal tissues and it was felt that this might support the view that the OKC (KCOT) had neoplastic potential. 28 Another study 29 showed similar results and suggested GP38 can be used to differentiate between the two varieties (parakeratinized and orthokeratinized). It has been suggested that whenever, there is loss of classic features of KCOTs due to inflammation this molecule may play a diagnostic role and differentiate aggressive (parakeratinized) and less aggressive (orthokeratinized) KCOTs.…”

3D imaging of human epidermis micromorphology by combining fluorescent dye, optical clearing and confocal microscopy

“…It is also known, like other dyes from the xanthenes family, to possess high fluorescent properties . In this work, we used it as a fluorochrome . Under UV light, the high intensity of fluorescent is detected in the epidermis.…”