MECHANISMS OF IRON II AND IRON III SEQUENCE HEMATOXYLIN STAINS

R. D. LILLIE ¹ and P. PIZZOLATO ¹

¹ Departments of Pathology, Louisiana State University School of Medicine and United States Veterans Administration Hospital, New Orleans, Louisiana 70112

Undifferentiated Fe II and Fe III hematoxylins yield blue to black nuclei, keratin, keratohyalin, enterochromaffin, Paneth and eosinophil leukocyte granules, erythrocytes, myelin and bile casts, gray to blue smooth and striated muscle, variable collagen and elastin staining and lighter gray cytoplasms. Staining is more intense with formol-free fixations. Staining is not prevented by acid hydrolysis which abolishes the Feulgen reaction and cationic dye staining. Acetylation and sulfation have little effect. Methylation and nitrous acid deamination sufficient to prevent cationic and anionic dye staining, respectively, prevent staining of erythrocytes, muscle, cytoplasm and zymogen granules but not that of nuclei, keratin, keratohyalin, trichohyalin, Paneth and eosinophil granules. These blockade effects are more complete with Fe II than Fe III. Saponification after methylation restores staining. With sublimate, Carnoy, alcohol and methanol-chloroform fixations, 2 N NaNO₂/HAc deaminates in 1 hr; with formol 18-24 hr or more may be required. The sites which stain with Fe II hematoxylin after adequate deamination have all given positive Sakaguchi reactions by -naphthol, 8-hydroxyquinoline and 2,4-dichloro-l-naphthol technics . The new -naphthoquinone-4-SO₃Na method also shows the same sites. The combined deamination, Fe II, hematoxylin technic merits further investigation as a possibly specific method not entailing hypochlorite or strong alkali and yielding stable blue to black stains of protein-bound arginine. Without deamination it appears that lysine, hydroxylysine and histidine and histamine should also react. Mast cells are often readily identified in blue or violet-black but are not found after deamination. Alkaline benzil, 9,10-phenanthrenequinone, 1,2-cyclohexanedione and glyoxal treatments irregularly weaken the deamination-resistant Fe II hematoxylin staining, though they do not prevent it. Low reactivity of hair cortex and coarse collagen bundles (requiring higher iron concentrations) is assigned to lower penetrability. Goblet cell, salivary gland, gastric and other mucins, pepsinogen granules, lipofuscins and cutaneous melanins do not stain with either Fe II or III sequence hematoxylins, either directly, after periodic acid oxidation, or after aminoisophthalic acid condensation at periodic acid-oxidized gastric mucin. Thus carboxylic, sulfuric and sulfonic acid and aldehyde sites are excluded. With the acylation effects, hydroxyl, tyrosyl and histidyl sites also appear to be excluded, and iron binding appears to be largely restricted to lysyl and arginyl sites. Impairment of the -naphthoquinone-4-SO₃Na-arginine reaction by prior iron binding supports the arginyl participation.

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