The widely used combination of hematoxylin and eosin allows the discrimination between nuclei and cytoplasm/connective tissue, respectively. Hematoxylin itself does not stain nuclei, but rather its oxidation product hematein. In this protocol, Meyer’s hematoxylin is used, which is chemically oxidized by sodium iodate. Hematein is anionic, as is nuclear chromatin, and will thus only stain nuclei in the presence of a mordant, in this case aluminium salt. The mordant/metal cation will positively charge hematein, providing a net charge able to bind and stain the anionic nucleic acids in chromatin in an acidic environment. The color change from red to blue occurs in alkaline pH, which is achieved by washing in a weak alkaline solution, such as tap water.

Eosin distinguishes the cytoplasm of different cell types and types of connective tissues by staining them different shades of red (Table 2). Fibrin is very acidophilic and is thus strongly stained by eosin and appears as distinctly pink (Fig. 1a, b) [11].

The MSB procedure includes application of Martius yellow, Brilliant Scarlet red, and Alcian blue dyes to easily distinguish erythrocyte cytoplasm, fibrin, and collagen, respectively, while nuclei appear black (Table 2, Fig. 1) [11]. Nuclei are first stained black with Weigert’s iron hematoxylin, a solution allowing stable and acid-resistant nuclear detection if compared to alum hematoxylins, such as Meyer’s hematoxylin. Martius yellow is then applied together with phosphotungstic acid in alcoholic solution to stain the cytoplasm of erythrocytes yellow. Mature fibrin will then be stained by brilliant crystal scarlet (Fig. 1c, d). Phosphotungstic acid will subsequently fix the dye in fibrin fibrils while removing any red stain from the collagen and blocking staining of muscle, collagen and connective tissue. Aniline blue subsequently stains collagen [11]. The MSB staining is selective for fibrin, but not entirely specific. Bone tissue, granulae in Paneth’s cells, and other cytoplasmic vesicles may stain. The combination of the three histological stains described in this chapter will resolve the detection of fibrin.

Perl’s iron visualizes ferric iron ions in hemosiderin, which is hydrated, protein-bound iron oxide (Fe(OH)₃). Hemosiderin is always located within cells and an intraplaque hemorrhage can result in hemosiderin deposits in the macrophages. In unstained or HE-stained tissue sections, hemosiderin presents as yellow-brown, coarse-grained pigment, which is more easily identified upon Perl’s Prussian blue staining [11]. Ferric iron in hemosiderin reacts with potassium ferrocyanide to yield insoluble blue ferric ferrocyanide precipitate (Prussian Blue, Fig. 1e, f). This reaction takes place in an acidic milieu to liberate iron from its protein carrier, such as hemoglobin, as follows: 4Fe(OH)₃ + 3K₄Fe(CN)₆ + 12HCl Fe₄[Fe(CN)₆]³ + 12KCl + 12H₂O [11].