Volume 24, Issue 4, Pages 267-274 (December 2017) A novel Golgi-Cox staining method for detecting and characterizing roles of the hepatic stellate cells in liver injury  Ma. de Jesús Gómez Villalobos, Susana Vidrio, Ruth Giles López, Gabriel Flores Gómez, Victoria Chagoya de Sánchez  Pathophysiology  Volume 24, Issue 4, Pages 267-274 (December 2017) DOI: 10.1016/j.pathophys.2017.06.003 Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 1 Morphological characteristics of hepatic lobule and hepatic stellate cell with Golgi-Cox staining of normal rat liver. Hepatic lobule was identified through the wide fiber network distributed in a radial form, from the central vein towards the periphery, generating their classical hexagonal shape (1A). Different amplifications of fiber network (1B and C). The network of fibers has wide (6–8μm) and fine fibers (<1μm), the fine fiber is distributed around the hepatocyte (1C, double stained-Golgi-Cox, and hematoxyline), whereas the wide fibers are distributed between the hepatocyte cords and from the central vein region toward the periphery of the hepatic lobule (1A, B). Fig. 1D shows one hepatic stellate cell from a normal rat liver impregnated with the Golgi-Cox stain, showing the classical stellate shape with the abundant and encircled fine cytoplasmic processes. CV: Central vein. Scale bar=thick black line. Pathophysiology 2017 24, 267-274DOI: (10.1016/j.pathophys.2017.06.003) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 2 Histological examination in normal and CCl4-damaged livers with Massońs Trichrome and Golgi-Cox stains. Liver section with Massońs Trichrome stain in normal livers had no evidence of fibrosis, with minimum smooth muscle cells around the central vein with typically hexagonal hepatic lobule architecture, and in accordance with the METAVIR score, all tissues were classified as 0 (2A). In Fig. 2B similar section tissue, but impregnated with Golgi-Cox stain, that also confirms the hepatic lobule architecture, but throughout the radial and abundant network of fiber, that cannot be observed with Massońs Trichrome stain. Tissue sections from CCl4-damaged livers and Massońs Trichrome stain classified with METAVIR score 1 or 2 show periportal fibrosis and narrow fibrous septum (2C). Tissue sections classified with METAVIR score 3 or 4 (2E) show infiltration and smooth muscle cells around the central vein with extensive periportal fibrosis, and frequent wide fibrous septum formation, generating a disruption of hepatic lobule architecture. Fig. 2D and F show tissue sections from CCl4-damaged livers impregnated with Golgi-Cox stain. The hepatic lobule architecture is lost, due to a significant reduction in the number of fine and wide fibers of the network. These changes were observed in tissues with a METAVIR score 1 or 2 (2D), is remarkable in tissues with a METAVIR score 3 or 4 (2F). As compared with the same section stained with trichrome stain, it is notable that Golgi-Cox stain is not able to stain fibrous septum. All sections in this figure are shown at the same amplification. CV: Central vein. Scale bar=thick black line. Pathophysiology 2017 24, 267-274DOI: (10.1016/j.pathophys.2017.06.003) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 3 The fine reticular fiber in the hepatic lobule in sections from normal and CCl4-damaged livers stained with reticulum stain. Fig. 3A and B show fine reticular fibers in the section from a normal liver in two amplifications. Reticular fibers are less than 1μm wide and are distributed around and between the hepatocyte. Fig. 3C and D show changes in the fibrillary network in sections from CCl4 livers damaged and METAVIR scores 3 or 4, also with two amplifications. Reticulum stain confirms the fibrous septa. CV: Central vein. Scale bar=thick black line. Pathophysiology 2017 24, 267-274DOI: (10.1016/j.pathophys.2017.06.003) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 4 Morphological characteristics of the hepatic stellate cell in normal and CCl4-damaged liver impregnated with the Golgi-Cox stain. Fig. 4A and B show hepatic stellate cells in the hepatic lobule from a normal liver with different amplification. HSCs have a stellar shape with a small size and multiple cytoplasmic processes encircled, around the capillaries, and distributed in zone one or the periportal region. Fig. 4C and D show the HSCs in CCl4-damaged liver sections with a METAVIR score 1 or 2. The HSCs are increased in size and density and distributed mainly in zone 3, instead of zone 1, as observed in normal liver sections. The soma shape is rounded and their cytoplasmic processes are wide and numerous. Fig. 4E and F show the hepatic stellate cells in CCl4-damaged liver sections with a METAVIR score of 3 or 4. Hepatic stellate cells increased in size, but a significant decrease in density was found in this stage. Fig. 4E shows an isolated hepatic stellate cell with an ovoid shape and wide and frayed appearance of cytoplasm processes. CV: Central vein. HSC: hepatic stellate cell. CV: Central vein. Scale bar=thick black line. Pathophysiology 2017 24, 267-274DOI: (10.1016/j.pathophys.2017.06.003) Copyright © 2017 Elsevier B.V. Terms and Conditions