Toward developing natural histologic stains using anthocyanins: A novel approach

Abstract

Introduction: In the present-day scenario, wherein histotechnological laboratory personnel come into contact with numerous hazardous chemicals every day, laboratories are emphasizing on development of safer and environment-friendly alternatives globally which are easily available and feasible. In this context, we have attempted to utilize anthocyanins, a family of pigments naturally occurring in fruits and flowers and which are already used as natural food colorants, for assessing their utility as histological stains.

Materials and methods: Juices of thin consistency from well-ripened pomegranates were obtained by using a juicer with blender functions. Formalin-fixed paraffin-embedded 4 μm thin sections were immersed in this solution for 2 h, during which the solution was periodically monitored. Several modifications were attempted such as the addition of Citrus limon (lemon) extract or acetic acid, change in orientation of the slides and refrigeration of the solution during the staining procedure.

Results: A peculiar pattern of reasonably diagnostic staining was observed in which the basal and suprabasal cells, basement membrane, inflammatory cells and collagen fibers stained prominently with a magenta color. The addition of lemon, horizontal orientation of slides and refrigeration each separately led to an improvement in staining characteristics. The addition of 4% acetic acid and refrigeration of the solution each led to an improvement in longevity of the staining solution.

Conclusion: Anthocyanins could potentially be used as viable histological stains having advantages of availability, feasibility, color stability and nontoxicity, although numerous modifications to improve longevity of staining solution and staining characteristics are warranted by further research for which the present study could serve as a pretext.

Keywords: Anthocyanins; coloring agents; flavonols; histologic technique; pomegranate; tissue stains.

Figure 1

Illustrates certain steps before beginning the staining procedure. (a) Edible part of the pomegranates utilized to prepare the staining solution. (b) Staining solution in Coplin jar. Note the red color of anthocyanin solution due to acidic pH. (c) Measurement of pH by a digital pH meter prior to commencement of staining procedure. (d) Staining solution filled in a container into which slides could be oriented horizontally during the staining procedure

Figure 2

Illustrates staining achieved by anthocyanin solution under. (a) Scanner view (×4). (b)×10 magnification. c) 40x magnification

Figure 3

(a) illustrates staining of connective tissue achieved by anthocyanin solution under scanner view. (b) Illustrates staining of connective tissue achieved by anthocyanin solution under ×10 magnification

Figure 4

Illustrates deposition of an aggregate at the bottom of the jar after storing the staining solution and leaving it undisturbed for more than 4 h