An adaptation of Twort’s method for polychromatic staining of epoxy-embedded semithin sections

Application of Several Special Staining Methods for Paraffin Sections on Epon-Embedded Semithin Sections

“…However, within the scope of staining methods applied for general or different purposes, there are studies that mention the staining results of tissue components in the present study. Therefore, the results of the present study are in agreement with the studies on light green and fast green FCF dyes that only stain type I collagen, especially trichrome techniques, and the identification of collagen and elastic fibers (29,31,36,41). We think that our study will be a guide in this field.…”

“…Another study reported that a mixture of azure B and basic fuchsin allowed the differentiation of many intra-and extracellular (collagen and elastic fibers) components in diverse colors and tones and that the method could also be useful for pathological tissues (40). Twort's staining method (neutral red and fast green FCF mixture) was adapted to epon-embedded sections in a study, which highlighted that in addition to many intracellular structures, extracellular collagen and elastic fibers could also be easily distinguished in many colors (41). A solution consisting of a toluidine blue and malachite green mixture and basic fuchsin for counterstaining was utilized in another study, in which discernible staining of nuclei, erythrocytes, mitochondria, collagen and elastic fibers, and cartilaginous structures was recorded (42).…”

Lung inflammation is associated with lipid deposition

“…Tissue specimens 2×2×2 mm in size were obtained from the inflamed site (in pneumonia specimens) or from intact lung tissue (in non-pneumonia specimens) fixed in 2.5% glutaraldehyde (Ted Pella, US) in cacodylate buffer, post-fixed in 1% OsO 4 , dehydrated in ethanol and propylene oxide (Sigma-Aldrich, US), and embedded in Epon (Fluka). We obtained semi-thin sections (0.5 μm thick) with a Leica ultramicrotome using glass knives and stained them on glass slides using methylene blue/azure II [11] or polychromatic Twort’s staining [12]. After washing in distilled water, the sections were air-dried and mounted in Epon resin under a coverslip.…”

“…Lung tissue specimens 1×1×1 mm in size were fixed in 2.5% glutaraldehyde (Ted Pella, US) in cacodylate buffer, post-fixed in 1% OsO 4 , dehydrated in ethanol and propylene oxide (Sigma-Aldrich, US), and embedded in Epon (Fluka, Switzerland). We obtained semi-thin sections (0.5 µm thick) with a Leica ultramicrotome using glass knives and stained them on glass slides using methylene blue/azure II [7] or polychromatic Twort’s staining, as described earlier [8] (Supplementary Figure S1A–B). The sections were mounted in Epon resin under a coverslip, and imaged using a Leica DM2000 microscope equipped with a Plan-Neofluar 100/1.3 Oil immersion objective and a Leica DFC7000 T camera.…”

In focus in HCB

“…Recently, Morikawa et al (2018) reported the use of an alkaline alcoholic solution of azure B and basic fuchsin as a polychromatic stain for semithin sections of double aldehydefixed and epoxy-embedded tissues. Manskikh and Sheval (2020) report now a simple, one-step polychromatic staining protocol using an aqueous alcoholic mixture of neutral red and fast green FCF, which is based on the histological stain introduced by Twort (1924). The adapted Twort's staining protocol was performed on semithin sections (0.25-0.5 μm thin) of glutaraldehyde-and osmium-fixed and epoxy resinembedded tissues, and does not require a pretreatment to remove the epoxy resin and osmium.…”