Histotechnology: Chapter 8

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Masson trichrome

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Most recipes produce red keratin and muscle fibers, blue or green collagen and bone, light red or pink cytoplasm, and dark brown to black cell nuclei.
The trichrome is applied by immersion of the fixated sample into Weigert's iron hematoxylin, and then three different solutions, labeled A, B, and C:
Weigert's hematoxylin is a sequence of three solutions: ferric chloride in diluted hydrochloric acid, hematoxylin in 95% ethanol, and potassium ferricyanide solution alkalized by sodium borate. It is used to stain the nuclei.
Solution A, also called plasma stain, contains acid fuchsin, Xylidine Ponceau, glacial acetic acid, and distilled water. Other red acid dyes can be used, e.g. the Biebrich scarlet in Lillie's trichrome.
Solution B contains phosphomolybdic acid in distilled water.
Solution C, also called fibre stain, contains Light Green SF yellowish, or alternatively Fast Green FCF. It is used to stain collagen. If blue is preferred to green, methyl blue, water blue or aniline blue can be substituted.

Front 2

Gomori one-step trichrome

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Purpose
To identify an increase in collagenous connective tissue fibers, or to differentiate between collagen and smooth muscle fibers
Principle
In the one-step trichrome procedure, a plasma stain (chromotrope 2R) and a connective tissue fiber stain (fast green FCF, light green, or aniline blue) are combined in a solution of phosphotungstic acid to which glacial acetic acid has been added. Phosphotungstic acid favors the red staining of muscle and cytoplasm. The tungstate ion is specifically taken up by collagen, and the connective tissue fiber stain is subsequently bound to this complex, coloring the collagen green or blue, depending on the counterstain used.

Front 3

van Gieson

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a mixture of Picric Acid and Acid Fuchsin. It is the simplest method of differential staining of Collagen and other Connective Tissue. 100 mL of Saturated Aqueous solution of Picric Acid added to 5 mL of 1% Aqueous solution of Acid Fuchsin.
The solution weakens after long standing and may be strengthened by adding few drops of fresh Acid Fuchsin. Nuclei - stains brownish black to black
Collagen (fibrous connective tissue) - stains pink or deep red
Muscle, Cytoplasm, RBC and Fibrin - stains yellow

Front 4

Verhoeff

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form a variety of cationic, anionic and non-ionic bonds with elastin, the main constituent of elastic fiber tissue[2]. Elastin has a strong affinity for the iron-hematoxylin complex formed by the reagents in the stain and will hence retain dye longer than other tissue elements. This allows elastin to remain stained, whilst remaining tissue elements are decolorized. Sodium thiosulfate is used to remove excess iodine and a counterstain (most often Van Gierson’s stain) is used to contrast the principal stain[4]. Elastic fibers and cell nuclei are stained black, collagen fibers are stained red and other tissue elements including cytoplasm are stained yellow.

Front 5

Gomori aldehyde fuchsin

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Classification: connective tissue stain

Mechanism of staining: ionic bonding/van der Waals forces

Purpose: stain elastin, beta cells in Islets of Langerhans, basophils in anterior pituitary, and mast cells

Control tissue: aorta, skin, lung, internal control
Elastin fibers: Royal purple

Muscle, cytoplasm, collagen: Green

Nuclei: Not demonstrated

Front 6

Russell modification of the Moval pentachrome stain

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intended for use in histological demonstration of collagen, elastin, muscle, mucin and fibrin in tissue sections. This procedure is particularly useful when studying the heart, blood vessels and various vascular diseases.

Elastic Fibers: Black to Blue/Black
Nuclei: Blue/Black
Collagen: Yellow
Reticular Fibers: Yellow
Mucin: Bright Blue
Fibrin: Bright Red
Muscle: Red

Front 7

silver impregnation for reticulum

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Front 8

Mallory phosphotungstic acid hematoxylin (PTAH)

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PTAH stains tissues either reddish brown or blue depending on their type. This property of simultaneously staining two different colours is different from other haematoxylin reagents e.g. alum-haematoxylin. The role of phosphotungstic acid and the mechanism of staining is not fully understood. Interestingly the active component of haematoxylin is the oxidised form, haematin, although this rarely acknowledged in the literature which refer to haematoxylin staining. Phosphotungstic acid forms a lake with haematin.[17] The make –up of the reagent is uncertain, examination of a year old sample showed there to be three coloured components, blue, red and yellow. These were not identified. Some investigations of “model” systems, reacting various compounds such as amino acids, purines, pyrimidines and amines with PTAH show that they give rise to different colours.

Front 9

methenamine- silver

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Front 10

oil red O

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a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance of a red powder with maximum absorption at 518 (359)nm.

Front 11

Sudan black B

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a nonfluorescent, relatively thermostable lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance of a dark brown to black powder with maximum absorption at 596-605 nm and melting point 120–124 °C. It stains blue-black.

Front 12

osmium tetroxide

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Front 13

toluidine blue

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Front 14

methyl green-pyronin

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Pyronin stains the cytoplasm of plasma cells and most nucleoli red. Methyl green stains DNA bluish-green.
Diagnostic Implications

May be useful in the identification of plasma cells and RNA in tissue sections and cytologic preparations.