Abstract
Although conventional toluidine blue staining is a common technique used for rapid observation of semithin sections prior to transmission electron microscopy, it is monochromatic and insufficient for accurate identification of different tissue components by light microscopy. Additionally, polychromatic staining methods generally require step-by-step processes involving different dyes, and it is often difficult to balance the color tone of each step. In this study, we developed a simple polychromatic staining method for epoxy-embedded tissue sections. We stained preheated sections with an aqueous ethanol solution of azure B and basic fuchsin, with the addition of sodium tetraborate to enhance the staining efficacy. We optimized various staining conditions to enable sufficient coloration easily and consistently in a single, rapid staining step, using a single staining-mixture solution. Our method enabled clear differentiation of various tissue structures according to color tone and stain intensity, thereby facilitating the detection of fine structural differences, including various organelle and inclusion bodies. This technique represents a simple polychrome-staining method to allow more informative and convincing histological investigation in various fields of research and education.
Highlights
Semithin sections of resin-embedded tissues are useful for identifying regions of interest that can subsequently be closely examined by transmission electron microscopy (TEM) and are considered a step in the preparation of TEM samples
Toluidine blue is a standard dye used for semithin sections [1,2] that allows for rapid assessment of regions of interest prior to TEM observation
Color differentiation was optimal at a dye ratio of 7:3 (0.05% azure B solution: 0.05% basic fuchsin solution)
Summary
Semithin sections of resin-embedded tissues are useful for identifying regions of interest that can subsequently be closely examined by transmission electron microscopy (TEM) and are considered a step in the preparation of TEM samples Due to their thinness (usually 0.5 μm) and the good morphological preservation achieved by treatment with reliable fixatives prepared for TEM, epoxy semithin sections are highly valuable, as they enable much finer visualization of tissue elements than. Various polychromatic staining techniques for epon-embedded semithin sections have been developed (Table 1) These methods usually require several staining steps and the preparation of numerous complicated reagents [3,4] or long incubation times [5,6,7]. It is possible that the colors may begin to fade within a few days;[5] the staining solutions may not remain stable and may need to be prepared fresh before each use [12,13]
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