Primo-Vascular System as Presented by Bong Han Kim

Table 1

Stains used by Bong Han Kim.

Stain	Target	Color	Mechanism	Reference
Feulgen	Cell nuclei, basophile granules, and other structures containing DNA inside p-subvessels. Basophile particles and p-microcells in sinuses of primo-node. Endothelial cell nuclei in walls of p-subvessels. DNA of p-microcell nucleosome.	DNA is stained red 570 nm. The background, if counterstained, is green.	Acid hydrolysis of DNA.	[6, 7, 37, 38]
Hillarp-Hokfelt	Chromaffin cells, epinephrine (adrenaline), and norepinephrine (noradrenaline). Small granules inside p-subvessels.	Yellow.	Oxidation of adrenaline and noradrenaline with potassium iodate gives a pigment formation. The method is based on the formation of iodochromes of the hormones.	[7, 39]
Sevki	Chromaffin cells, epinephrine, and norepinephrine.	Chromaffin cells: bluish-red; inside primo-node: red to bluish-red; red blood cells: brown; mast cell granules: red.	Sevki stain (~6% water dilution of Giemsa stain).	[7, 40–42]
Giemsa	Adrenal medullary cells, chromaffin cells, epinephrine (adrenaline), norepinephrine (noradrenaline), collagen, erythrocytes, platelets, lymphocytes, monocytes, megakaryocytes, and hemocytoblasts.	Chromaffin: brown; collagen: blue; erythrocytes: pink; platelets: light pale pink; lymphocyte: sky blue; monocyte: pale blue; leukocyte nuclear chromatin: magenta; megakaryocytes: reddish-blue nuclei and blue cytoplasm; hemocytoblasts: large, vesicular, pink nuclei, and prominent nucleoli. Giemsa, after a dichromate fixation, produced a green color.	Giemsa’s solution is a mixture of methylene blue, eosin, and Azure B. Methylene blue is a cationic dye. It binds to tissue anions and stains basophilic substances, including nucleic acids. Eosin is an anionic dye and is attracted to positively charged protein groups (cations), such as amino groups. It is an acidophilic stain. Azure B is formed by the oxidation of methylene blue and is a basic stain.	[7, 40, 43–45]
Gros-Schultze	Nerve fibers and nerve endings.	Dark brown to black.	Silver nitrate bath. The silver nitrate is reduced by the sodium potassium tartrate into a metal silver that is adsorbed by argyrophilic nerve fibers.	[7, 46, 47]
Van Gieson	Neural vessels. Collagen.	Collagen: pink or deep red; cytoplasm: yellow; elastic fibers: blue to black.	Mixture of picric acid and acid fuchsine (picrofuchsin). The method is based on the affinity towards elastic fibers displayed by the dye resulting from a reaction between resorcin and basic fuchsine in the presence of ferric chloride.	[7, 43, 48]
Verhoeff	Elastic fibers, nuclei, and collagen.	Elastic fibers: intense blue-black to black; nuclei: blue to black; collagen: red; other: yellow. Eosin counterstain shows erythrocytes red.	^aCombination of stains: hematoxylin, iron(III) chloride, Lugol’s iodine, Van Gieson’s stain (acid fuchsine, picric acid), and sodium thiosulfate. The tissue is stained with a hematoxylin, ferric chloride, and iodine.	[7, 49, 50]
Unna-Pappenheim	RNA and DNA in tissue sections.	RNA: red; DNA: green.	^abMethyl green-pyronin combination. Competition between the slow staining, but doubly charged, methyl green and the more rapidly staining, singly charged pyronin Y.	[6, 51, 52]
Brachet	DNA, RNA, and DNA in the nucleus of cells and RNA in the nucleolus. RNA in the cytoplasm of cells. DNA in p-microcell nucleoplasm.	DNA: green; RNA; p-microcell nucleoplasm: Red.	Methyl green-pyronin combination. Similar to that of Unna-Pappenheim method. Brachet introduced control by using RNase solution before staining.	[7, 50, 51]
Acridine-orange	Vital stain for DNA and RNA. Primo-vascular nodes and vessels. Acridine orange also accumulates and emits red light in mast secretory granules and other cellular acidic compartments.	RNA: fluorescent red; DNA: fluorescent green; mast cells: red.	Acridine orange is a basic dye. Basic dyes are cationic and will stain anionic or acidic molecules. Staining is pH sensitive. Acidic substances that stain with basic dyes are termed basophilic.	[6, 51, 53, 54]
Hematoxylin-eosin	The basophilic structures containing nucleic acids, such as the ribosomes and the chromatin-rich cell nucleus, and the cytoplasmic regions rich in RNA. The eosinophilic structures composed of intracellular or extracellular protein. Most of the cytoplasm is eosinophilic.	Hematoxylin colors basophilic structures with blue-purple hue and alcohol-based acidic eosin colors eosinophilic structures as bright pink. Red blood cells, collagen fiber: red.	Oxidized hematoxylin (hematein) has a selective affinity for nuclei when combined with aluminum ion. The mechanism of eosin staining is not fully understood but is believed to be of an electrostatic nature. Negatively charged eosin ions will stain positively charged tissue ions.	[7, 43, 50, 55]
Resorcin-fuchsin	Glycogen, basement membrane, reticulum fibers, collagen, and other structures containing polysaccharides. flexible fibers, and collagen inside primo-nodes.	Elastic fiber: purple; elastic membrane in blood vessel: dark purple; nuclei: pale red; red blood cells: red if counterstained by eosin.	Acetylation, sulfaction, and phosphorylation induce binding of resorcin-fuchsin.	[7, 43, 56]

Verhoeff mechanism: the differentiating is accomplished by using excess of ferric chloride to break the tissue-ferric chloride dye complex. The dye will be attracted to the larger amount of ferric chloride in the differentiating solution and will be removed from the tissue. The elastic fibers have the strongest affinity of the iron-hematoxylin complex and will retain the dye longer than the other tissue components. Van Gieson’s solution is used as a counterstain. ^bUnna-Pappenheim mechanism: methyl green has two cationic charged groups that become linked to the phosphate moieties in the DNA. The pyronin Y displaces the methyl green from all sites of linkage except where its double charge gives it a selective advantage (acidic polymer such as DNA). Consequently the methyl green stains DNA and retains its binding to this substance against the competitive action of pyronin Y. Pyronin Y stains the less polymerized RNA rapidly, and it can displace methyl green from linkages having smaller polymeric acidic substances (RNA).