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Background: Three-dimensional (3D) reconstruction of human peripheral nerves, as a useful tool to understand the nerve internal information and functional basis, has become an important area of research in the peripheral nerve field. Methods: In this study, we proposed a two-dimensional (2D) Karnovsky-Roots toluidine blue ponceau 2R (K-B-2R) staining method based upon conventional Karnovsky-Roots staining. It significantly improved the ability to display nerve fascicles, motor and sensory nerve fiber textures. In this method, Karnovsky-Roots staining was carried out, followed by toluidine blue counterstain and ponceau 2R counterstain. Results: Comparisons were conducted between the three methods in staining of median nerve sections, which showed similar distribution characters in acetylcholinesterase-positive sites. The additional counterstaining did not change the basis of Karnovsky-Roots staining. However, the resulting images from this new method significantly facilitated the subsequent 3D nerve reconstruction and 3D printing. Conclusions: These results show that the new staining method significantly enhanced the display qualities of nerve fascicle edges and fiber textures of motor and sensory nerves and facilitated 3D nerve reconstruction. usc Refereed/Peer-reviewed

Although collagen of either tendon or dermis can be stained equally well with Ponceau 2R/Acid Fuchsin or Light Green SF if the dyes are used independently, tendon collagen retains the red dye mixture and dermal collagen the green counterstain when the dyes are used sequentially in the Masson trichrome procedure. The results of experiments designed to assess differences in the penetration, retention and displacement of these arylmethane dyes have demonstrated that they are retained more firmly by the tensioned collagen of tendon or stretched dermis, and are more easily displaced from the collagen of relaxed tendon or dermis.

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified Verhoeff's elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by lissamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsin, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.

Although normally, dermal collagen fails to retain the Ponceau 2R/Acid Fuchsin component of the Masson trichrome procedure and is stained green by the counterstain, the collagen of sections of human dermis which have been heated above its denaturation temperature (Ts) in moist conditions will retain the initial red stain. This difference in dye retention appears to be related to conformational changes of the collagen molecule which are associated with denaturation, for the change in staining is directly related to the shrinkage temperature, is reversed by rapid cooling after heating, is profoundly affected by differences in environmental conditions during heating which affect molecular rearrangement, and is age-dependent.

Several dyes, notably ponceau 2R, azofuchsin 3B, nitrazine yellow, and Biebrich scarlet may replace imported “ponceau de xylidin” in the Masson ponceau acid fuchsin mixture. Of these Biebrich scarlet appears to be the best and may be used without acid fuchsin.A mixture of equal parts of 5% solutions of phosphomolybdic and phosphotungstic acids is much superior to either acid alone and gives adequate mordanting in 1 minute at 22°C.With the fast green modification, times in plasma and fiber stains can be reduced to 2 minutes each. With anilin blue a 4-minute plasma stain is required. One-minute final differentiation in 1% acetic acid is adequate.Primary mordanting of formalin material may be accomplished by 5 minutes in saturated aqueous mercuric chloride or 2 minutes in saturated alcoholic picric acid. Three minutes washing in running water is required after these mordants.

The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H2SO4 per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable...