Special Stains Issue 1 April 2010 B A Figure 1. Fatty liver metamorphosis. A) Iron, 20x; B) H&E, 40x. 40x. 40x. 40x. 20x; B) H&E, 20x; B) H&E, 20x; B) H&E, Iron, Iron, Iron, A) A) A) metamorphosis. metamorphosis. metamorphosis. liver liver liver 1. Fatty 1. Fatty 1. Fatty Figure Figure Figure A new look some old favorites at Iron is normally complex, an iron-storage Hemosiderin, However, intracellularlypresent in macrophages. during hemorrhaging, when red blood cells (RBC) are the circulatory from excess hemosid- released system, erin deposits will extracel occur in the surrounding - This is seen grossly in the color of change lular spaces. a bruise.The initial blue-red color of a bruise indicates of blood the blood due to tis- the release from vessel sue trauma.The macrophages then engulf the RBC (phagocytosis) and degrade the hemoglobin into then bilirubin and finally (yellow) (green), biliverdin An accumulation into hemosiderin (yellow-brown). of hemosiderin deposits in tissue also occurs in iron On an H&E overload (hemosiderosis). disorders crystal to brown appears as yellow preparation iron - line deposits between or within cells (Figure 1-B). Blue (Figure Prussian 1-A) can be used to more Perl’s in hemo- iron The easily demonstrate in tissues. iron and siderin +3) is complexed to a protein (ferric ion; cannot Exposing chemically the hemosiderin react. to a solution of hydrochloric acid releases the ferric with potassium ferrocyanideion allowing it to react to ferric ferrocyanide. form precipitate, blue the Prussian While we refer to this as it a isspecial actually“stain” a histochemical demonstration since the colored of iron substance formed is not a dye. INNOVATOR -

- of provid tradition with ANATECH’s In keeping our through information technical ing complete discusses issue of the Innovator this newsletter, family of the chemistry and uses of ANATECH’s special stains. We hope you“solutions”! our enjoy reading about ANATECH LTD. is pleased to announce that our our that announce is pleased to LTD. ANATECH in kits as available now special stains are really well as individual units. as pH 2.5 as well Blue, a kit Alcian offer for we For staining of mucin, stain also package complete We Mucicarmine. and our popular Hp Iron, Blue kits Amyloid, for . Each of these kits Helicobacter for Yellow and Hp stain your to required all the reagents contain tissue sections along with a detailed product in- sert to provide step-by-stepperform to each stain. instructions on how special special them as really to refer We special stains. unique and were of them are because several with the a problem to in response developed existing traditional stain, the By understanding performance. or technical due to unavailability able to was LTD. ANATECH chemistry of dyes, special and produce these problems to respond chemically unique and/or offer are stains that the conventional over improvement a technical appear the stained tissue’s that stain. Knowing our really is critical, ance special stains yield dyes. as the traditional results similarly colored ANATECH LTD. has a growing family of really has a growing LTD. ANATECH Hematoxylin and eosin (H & E) is the gold stan- and eosin (H & E) is the Hematoxylin of tissue structure in demonstration for dard - utilizing vari by However, pathology. anatomic further allow special stains solutions, ous dye visualization of major macromoleculescarbohydrates, (e.g.,proteins, minerals) the blue and pink hues of H & E beyond of colors in a rainbow special stains indispensable makes This staining. in the demonstration of tissueits components. morphology While and immunohistochemistry in truly advancing and molecular biology are the diagnosis of diseases, them special stains makes of histochemical cost the comparatively low laboratory. the pathology vital to Special Stains Issue Stains Special ANATECH LTD ANATECH - - H) and carboxyl (COOH) (COOH) H) and carboxyl 3 groups (blue) of sulfate (blue) of sulfate groups (SO Figure 2. Chondroitin 2. Chondroitin Figure A is an acid muco sulfate acidic The polysaccharide. classify it as an acidic mu copolysaccharide. Figure 3. ANATECH’s Alcian Blue, pH 2.5 and Brazilliant!. Colon, 10x. 10x. 10x. Colon, Colon, Colon, pH 2.5 and Brazilliant!. pH 2.5 and Brazilliant!. pH 2.5 and Brazilliant!. Blue, Blue, Blue, Alcian Alcian Alcian 3. ANATECH’s 3. ANATECH’s 3. ANATECH’s Figure Figure Figure In 1970, due to safety and environmental concerns, due to safety concerns, and environmental In 1970, Imperial Chemical United Kingdom) Industries (ICI, stopped production of Alcian blue. largeHowever, stocks available of their batch numbers 8GS and 8GX were worldwide until 2001 when the inventory was finally exhausted. Determined to replenish this important sought a safer manufactur- ANATECH diagnostic stain, was a dependresult - The ing method dye. to obtain the able supply certified blue dye of Alcian by the Biological has been instrumental ANATECH Stain Commission. The suf- in filling the pipeline with Alcian blue since! ever used no longer because 8GS and 8GX are theywere fixes expressly an ICI manufacturing identification. another stain used to demonstrate acid Mucicarmine, mucopolysaccharides, is an aqueous solution comprised of carmine and aluminum chloride. Carmine dye has story interesting its own bugs! Female from made – it’s dried harvested are cochineal nopal cactus, insects, from isolated and Carminic acid, into a powder. and ground purifiedby extracting the cochineal with water powder is then combined with a metal such- as alumi or alcohol, num to form the dye. Carmine dye and the subsequent - - - Anatech Mucin Mucin belongs to the carbohydrate family of molecules known as mucopolysaccharides, glycoaminoglycans or Mucopolysaccharides of a chain made glycoproteins. are of repeating sugars with side chains containing amino and/or acidic elements.The presence or absence of an acidic side chain determines whether the mucopolysac charide is classified as an acid or neutral mucopolysac Figureof the repeating unit 2 shows respectively. charide, chondroitin sulfate, an acid mucopolysaccharide that is Mucosubstances are found in cartilage substance. ground normally found in connective tissues and epithelial cells An overproduction of the lung and intestine (Figure 3). of mucin is indicative of disease. Alcian blue stains acid mucopolysaccharides and is useful in identifying mucin secreting adenocarcinomas or in the diagnosis of Barrett’s esophagus. Barrett’s esophagus, a the normalcondition stratified where squamous epithe- lium of the esophagus is replaced with the metaplastic simple columnar type will have the small of intestine, goblet cells containing acid mucopolysaccharides. is positivelyAlcian blue dye charged (cationic) and binds to the negatively charged (anionic) carboxyl or sulfate groups of acid mucopolysaccharides.The result ing complex is a very stable salt and generally cannot be stain solutionThe pH of the Alcian blue can de-stained. be varied to distinguish component the acid (carboxyl or sulfate). At pH 2.5 both carboxylated and sulfated acids At pH 1.0 only the sulfated with Alcian blue. will react or as seen in carcinoma of the prostate willacids react, malignant synovioma. Innovator

Special Stains Issue 2 April 2010 Special Stains Issue 3 April 2010 are difficult to difficult are are now recognized as the bacteria that Anatech Innovator A B Figure 6. ANATECH’s Amyloid Red and Harris Hematoxylin. Red Red Red and Harris Hematoxylin. and Harris Hematoxylin. and Harris Hematoxylin. Amyloid Amyloid Amyloid 6. ANATECH’s 6. ANATECH’s 6. ANATECH’s Figure Figure Figure 40x. 40x. Kidney, Kidney, Figure 5. Amyloid Red’s flat structure (A) lays in the grooves of the of the of the of the in the grooves in the grooves in the grooves lays lays lays structure structure structure flat (A) flat (A) flat (A) Red’s Red’s Red’s 5. Amyloid 5. Amyloid 5. Amyloid Figure Figure Figure (B). (B). molecule molecule protein protein beta-pleated beta-pleated Helicobacter pylori for responsible most stomach ulcers and gastritis. are Enzymes pylori and cytotoxinsbreak H. by produced of the stomach lining cells mucin the protective down (mucous cap cells), directly exposing the stomach to acid Helicobacterand causing inflammation. see microscopically because they often are embedded in the mucus. High-contrast colors, as produced by the were classified by OSHA as known human carcinogens. classified human as known by OSHA were carcinogens. At that time, introduced ANATECH Amyloid Red, a dye with the required chemical structure (Figure 5) that is Amyloid Red (Figure- 6) pro not a benzidene derivative. vides the same selectivity for while amyloid red as Congo the histology from a carcinogen removing lab. Helicobacter - Figure 4. ANATECH’s EZ Mucicarmine and EZ Green. Colon, 20x. 20x. 20x. 20x. Colon, Colon, Colon, Colon, EZ Mucicarmine and EZ Green. EZ Mucicarmine and EZ Green. EZ Mucicarmine and EZ Green. EZ Mucicarmine and EZ Green. 4. ANATECH’s 4. ANATECH’s 4. ANATECH’s 4. ANATECH’s Figure Figure Figure Figure Amyloid is a protein that disrupts the normal physiology These deposits may be dispersed of tissue when deposited. the bodythroughout (systemic amyloidosis) or can be organ specific heart). liver, kidney, Amyloid (e.g., plaques diseases, associated neurodegenerative many with are such as Alzheimer’sThe andmost Parkinson’s. common used to demonstrate amyloiddye is the benzidene dye, long flat structure fits perfectly red’s Congo red. Congo into the grooves of amyloid’s pleated fibril proteins. In addition Congo red has the required hydrogen atom perfectly that aligns spacing of amyloid with the proteins to allow dye-tissue bonding. In 2000, benzidene dyes Amyloid ANATECH’s higher-grade carmine dye powder is avail carmine higher-grade - powder dye ANATECH’s able for those who want to make their own mucicarmine eliminating the need readily water-soluble, It’s solution. - the addi However, the stain. for boiling water to prepare tion of the aluminum chloride to the solution generates hydrochloric acid vapors and therefore must be handled eliminate this hazard for To under a chemical fume hood. EZ Mucicar offers- LTD. ANATECH the histology lab, mine, a ready to use mucicarmine solution. Our Mu cicarmine kit contains EZ Mucicarmine, Weigert iron iron Weigert cicarmine Mucicarmine, kit contains EZ hematoxylin for nuclear staining along with EZ Green, a cytoplasmicgreen counterstain (Figure 4). mucicarmine solutions have shown variability in staining In 2005 the BiologicalStain - Commis years. for many sion reported a drop in the dye content of carmine.This was attributed to variability in the insects themselves as well the processing as the extraction used by procedures market a car to - brought In 2007 ANATECH plants. a South by American manufactured mine dye company of insect collec process - the entire over control that had tion and chemical manufacturing.This total control has eliminated the inconsistency lots and produced of past higher-grade carmine. 2-500ml 1-500ml 1-500ml 1-500ml 1-500ml 2-500ml 2-500ml 1-500ml 1-500ml 1-250ml 1-250ml 1-500ml 1-500ml 1-500ml 1-250ml 1-250ml 1020 Harts Road Lake MI 49037 Creek Battle 1.800.262.8324 www.anatechltdusa.com Alcian Blue, pH 2.5 Stain Kit pH 2.5 Stain Blue, Alcian acid 3% Acetic pH 2.5 Alcian Blue, Brazilliant! Kit Stain Amyloid Amyloid Red Stain Harris Hematoxylin Kit Stain Helicobacter acid 1% Periodic acid 10% Acetic Hp Blue Yellow Hp kit Stain Iron ferrocyanide 2% Potassium acid 4% Hydrochloric Brazilliant! Kit Mucicarmine Stain Green EZ MucicarmineEZ Solution A Weigert Solution B Weigert Kit contains: Kit contains: Kit contains: Kit contains: Kit contains: Special Stain kits available kits available Stain Special #880 Cat Alcian Blue, pH 2.5 solution stains acid mucopolysaccharides while Brazilliant! blue aqua stains nuclei red. #881 Cat Amyloid Red solution stains amyloid deposits red and Harris hematoxylin stains nuclei blue. #882 Cat mucin stains Yellow blue and Hp pylori Hp Blue stains H. yellow-green. #883 Cat The working iron solution reacts with iron deposits forming a Brazilliant! stains nuclei blue). blue pigment (Prussian red. #884 Cat EZ Mucicarmine stains mucins a bright reddish-pink, hematoxylinWeigert stains nuclei black, and EZ Green gives counterstain. a green - - Anatech ANATECH, LTD. Three Dyes, Three Dilemmas. The The Dilemmas. Three Dyes, Three LTD. ANATECH, 2001. Autumn Innovator, The Helicobacter. for Searching LTD. ANATECH, Spring 2002. Innovator, Job Your Mucicarmine Makes New ANATECH,LTD. 2007. Winter Innovator, The EZ. Blue- Alcian The Ugly Stain: The Wendi. Edgett, Esophageal Biop for Stain Yellow H&E-Metanil sies. Mikro-Graf. 32(4): 1, 4-5: December 2003. 32(4): 1, 4-5: December Mikro-Graf. sies. and Histochemical A. Histological Kiernan, J. Butterworth-Heine Woburn: ed. 3rd Methods. mann, 1999. - Basic Pathol Marcia. Stanley; Angell, Robbins, 1976. Saunders, W.B. Philadelphia: 2nd ed. ogy. http:// from: Hemosiderin. Available on en.wikipedia.org/wiki/Hemosiderin. Accessed 3, 2010. February Histochemistry. Diagnostic Frederick. Zugibe, 1970. Co., Mosby C.V. Saint Louis: Figure 7. ANATECH’s Hp Blue and Hp Yellow. Stomach, 100x. 100x. 100x. 100x. Stomach, Stomach, Stomach, Stomach, Yellow. Yellow. Yellow. Yellow. Hp Hp Hp Hp Blue and Hp Blue and Hp Blue and Hp Blue and Hp 7. ANATECH’s 7. ANATECH’s 7. ANATECH’s 7. ANATECH’s Figure Figure Figure Figure Innovator References: • • • • • • • • Alcian yellow (mucus) and toluidine blue (dark blue make it easier to detect the bacteria) procedure, bacteria.The unavailability of Alcianyellow in 2002 to develop an alternative ANATECH prompted the to replace Yellow) (Hp Blue and Hp procedure traditional method.Hp Blue ANATECH’s stains the bacteria blue and stains HpYellow the mucus a contrasting yellow-green (Figure 7). Although Alcian yellow has returned to the market its price makes it is fast and Yellow Hp Blue and Hp cost-prohibitive. economical to other procedures. when compared

Special Stains Issue 4 April 2010