Histopath Proteins and Nucleic Acids Flashcards by ANTOINE GABRIELE Z. JUMAGBAS

is the basic component of living cells and
is made of carbon, hydrogen, oxygen, nitrogen
and one or more chains of amino acids linked by
peptide bonds.

Protein

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Proteins in Tissues
Proteins based on PHYSICAL
CONFIGURATION

Fibrous proteins
● Globular proteins
● Membrane proteins

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Proteins in Tissues
Proteins based on CHEMICAL COMPOSITION (3)

Simple proteins
● Conjugated proteins
● Derived proteins

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Proteins based on CHEMICAL COMPOSITION

o On hydrolysis they yield only the amino acids
and occasional small carbohydrate
compounds.

o E.g albumins, globulins, structural proteins,
enzymes, histones and protamines

Simple Proteins

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Proteins based on CHEMICAL COMPOSITION

o These are proteins derived from simple or
conjugated proteins by physical or chemical
means

o E.g denatured proteins and peptides

Derived Proteins

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Proteins based on PHYSICAL CONFIGURATION

o Play several roles including relaying signals
within cells, allowing cells to interact, and
transporting molecules.
o E.g. c-myc, estrogen receptor, glycophorin D,
histones, hydrolases, oxidoreductases, and p53

Membrane Proteins

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Proteins based on CHEMICAL COMPOSITION

o These are simple proteins combined with
some non-protein material in the body to form
complex proteins.

o E.g lipoproteins, mucoproteins, nucleoproteins,
glycoproteins, and phosphoproteins.

Conjugated Proteins

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Proteins based on PHYSICAL CONFIGURATION

o Are more water soluble than the other classes of proteins and they have several functions
including transporting, catalyzing, and
regulating.

o E.g. albumins, alpha globulin, beta globulin, fibrin,
gamma globulin, hemoglobin immunoglobulin and

Globular Proteins

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Histochemical Identification of Proteins

Most commonly used fixative for amino
acid histochemistry

Neutral Buffered Formol Saline

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Proteins based on PHYSICAL CONFIGURATION

o Form muscle fiber, tendons, connective tissue
and bone. Fibrous proteins can be demonstrated
by selective staining with small or large molecule dyes (trichome method), and silver
impregnation (reticulin methods), and specific
dye-protein interactions

o E.g. actin, collagen, elastin, fibronectin, myosin,
tau, tropomyosin and tubulin.

Fibrous Proteins

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Staining depends largely on the attachment of
dyes to proteins that have both positively and
negatively charged groups Phosphate groups of
DNA also are important in nuclear staining. A
tissue section contains many proteins that differ
in their isoelectric points. At an ideal pH, certain
tissue components will show a relative acidophilia
whereas others display a relative basophilia.

Principles of Staining

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Histochemical Identification of Proteins

Histochemical methods are used to demonstrate
the ______________________________________rather
than whole protein molecules. They are based
upon identification of specific linkages or groups
within the amino acid molecule

presence of amino acid molecules

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are attracted to acidic substances, such as mitochondria and collagen which are anionic (negatively charged) at physiologic pH. Many proteins are acidophilic at physiologic pH. The aniline dye, eosin, is an acid
dye that stains cytoplasm, muscle, and
connective tissues in various shades of pink and
orange. Eosin is a red or pink that is
Acidic/Negative

Acidophilic dyes

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Ways of sharpening nuclear staining
○ Adding a ___________amount of____________
to ______ aqueous solutions of neutral red
○ Final pH usually ____
○ Addition of borax to _________________________

very small, acetic acid, 1-2%, pH 4, Methylene Blue

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are attracted to basic
substances, which are cationic (positively
charged) at physiologic pH. Proteins are
basophilic at a pH lower (more acidic) than their
isoelectric point. When the environmental pH is
below a protein’s isoelectric point, the protein is
negatively charged and hence basophilic

Basophilic dyes

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ACID loves _____; BASE loves _____

BASE
ACID

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Attracted to basic substances (cytoplasm,
muscle, connective tissue, colloid, red
blood cells, decalcified bone matrix)

Acidic Dyes (Basophilic)

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Attracted to acidic substances
(mitochondria, collagen)

Basic Dyes (Acidophilic)

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Histochemical Identification of Proteins

Reacts with amino acid groups

Avoid MERCURIC CHLORIDE as fixative

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Fast Green is an acid dye that stains basic
groups in the tissues, particularly basic
protamines and histones which have higher
isoelectrical points than the pH of the staining
solution. All other proteins have lower isoelectric
points: their basic groups are not ionized and
therefore will not stain

Alkaline Fast-Green Method for Basic Proteins

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Peracetic Acid oxidizes cystine and cysteine,
forming strong cysteic acid which is stained
blue-green by a basic dye. Sakaguchi’s test for
arginine used NaOH, sodium hypochlorite
(Milton’s reagent) and pyridine chloroform,
producing orange-red color on objects containing
arginine. The mechanism is not known.

Peracetic Acid-Alcian Blue for Cystine and
Cysteine

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Peracetic Acid-Alcian Blue for Cystine and
Cysteine covalently attached glycosaminoglycan chain(s). The chains are long , linear carbohydrate
polymers that are negatively charged under
physiological conditions due to the occurrence of
sulfate and uronic acid groups

Proteoglycans

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This is a combined method utilizing the
properties of both the PAS and Alcian blue
methods to demonstrate the full complement of
tissue proteoglycans. The rationale of the
technique is that by first staining all the acidic
mucins with Alcian blue, those remaining acidic
mucins which are also PAS positive will be
chemically blocked and will not react further
during the technique.

Alcian Blue - PAS Staining for Proteoglycans

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Demonstration of nucleic acids depends upon
either reaction of the dyes with the phosphate
groups, or production of aldehydes from the
sugar (deoxyribose). No histochemical methods
are available to demonstrate the nitrogenous
base

STAINING OF NUCLEIC ACIDS

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STAINING OF NUCLEIC ACIDS demonstrate sugar

Feulgen technique -

STAINING OF NUCLEIC ACIDS demonstrates phosphate

Methy green pyronin technique

STAINING OF NUCLEIC ACIDS demonstrates both DNA and RNA

Gallocyanin-chrome alum method

uses the basic dyes to produce a differential staining reaction for DNA and RNA respectively. Methy green is highly selective for DNA, coloring it green due to the binding of anionic phosphate group with the stain. Pyronin is somewhat specific for RNA, giving it a pinkish red color. Methy green-pyronin stain is also utilized to detect the presence of plasma cells and lymphocytes

Methyl Green - Pyronin method for RNA and DNA

Methyl Green - Pyronin method for RNA and DNA DNA (chromatin): ___________________ RNA (Nucleoli): ________ Granule: _______________ Plasma cell cytoplasm: _______

Green or blue-green Rose-red Dark rose-red Purple

Feulgen Staining for Nuclear DNA DNA - ____________ Cytoplasm - ______

red purple, green

is a staining technique used to identify chromosomal material or DNA in cell specimens. Acid hydrolysis removes purine bases from the DNA, thereby unmasking free aldehyde groups. Feulgen reaction allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with the free aldehyde groups in proportion to the DNA concentration in the cell which results in the purple staining.

Feulgen Staining for Nuclear DNA

are fluorescent dyes which emit light or visible radiation energy when excited by light of shorter wavelength, either visible or ultraviolet

Fluorochromes

FLUOROCHROME TRACERS (4)

○ Fluorescein ○ Rhodamine ○ Acridine Orange ○ Acriflavine

Fluorochrome Traces still the most widely used fluorochrome, because of its wide absorption spectrum and blue light range

Fluorescein

Fluorochrome Traces conjugates absorb maximally in green light, exhibiting an orange-red emission, and are commonly used in two-color techniques

Rhodamine

Fluorochrome Traces the most commonly used fluorochrome to demonstrate DNA and RNA in fresh or fixed tissues

Acridine Orange

CONNECTIVE TISSUES (8)

1. Loose connective tissue 2. Adipose Tissue 3. Dense Connective Tissue 4. Cartilage 5. Bone Tissue 6. Reticulin (Reticular Connective Tissue) 7. Elastic Tissue 8. Blood Plasma

Fluorochrome Traces Can be used as an alternative to basic fuchsin in Schiff’s reagent. DNA is stained by a fluorescent yellow color in this Feulgen-type reaction

Acriflavine

constitutes a nonliving framework within the various organs, and is made up of various cell components that are found in between other tissues everywhere in the body. Its one of the four types of biological tissue that support, connect, or separate different types of tissues and organs in the body.

Connective tissue

RETICULIN STAIN techniques for staining of reticulin depend on local reduction and selective precipitation of silver by the carbohydrate aldehyde groups of reticulin

Silver impregnation t

Gomori's Silver Impregnation Stain for Reticulin are silver stains based on the argyrophilic properties of reticulin fibers. Reticulin fibres have little natural affinity for silver solutions so, they must be treated with a suitable solution, (2.5% iron alum) and potassium permanganate to sensitize areas within the fibres where silver deposition can be initiated.

Reticulin stains

RETICULIN STAIN is are commonly demonstrated by the use of stains involving silver solutions These stains rely on the impregnation of silver ions to the fibers and subsequent reduction of those silver lons to their visible metallic form.

Reticular fibers

Reticulin stains results

Reticulin fibers black

In this method, the tissue is oxidized by potassium permanganate to enhance subsequent staining of reticular fibers. The excess permanganate solution is removed by oxalic acid. The iron alum serves as sensitizer and is subsequently replaced by silver solution to form silver oxide

Reticulin Stain -Gordon and Sweets' Method (1936)

Reticulin Stain -Gordon and Sweets' Method (1936) Results: ○ Reticulin fibers = ○ Nuclei = ○ Background =

Results: ○ Black ○ Black ○ Red

forms a coarser extracellular framework than reticulin. Collagenous fibers are found in ligaments, tendons, cartilage and bone. Its doubly refractile, coarse connective tissue fibers stain yellow, lavender or brown on silver impregnation, and red with van Gieson's stain.

● Collagen

The most commonly used acid is ___________, which also acts as a _______________ for muscle and cytoplasm.

picric acid counterstain

Collagen may be differentially stained by any of the following techniques: (5)

○ Van Gieson's stain ○ Masson's Trichrome stain ○ Mallory's Aniline Blue stain ○ Azocarmine stain ○ Krajian's Aniline Blue stain

is the simplest method of differential staining of collagen and other connective tissue that uses a mixture of picric acid and acid fuchsin. In van Gieson stained preparations collagen stains dark red while other tissue components appear in varying shades of grey (nuclei) and yellow (cytoplasm).

Van Gieson's Stain for Collagen (Carson 2009)

is a one-step method where 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.

GOMORI'S ONE-STEP TRICHROME STAIN

Van Gieson's Stain for Collagen (Carson 2009) Results: ○ Nuclei = ○ Collagen (fibrous connective tissue) = ○ Muscle, Cytoplasm, RBC and Fibrin =

brownish black to black pink or deep red Yellow

This is often used to stain connective tissue. Trichrome means the technique produces three colors. Nuclei and other basophilic structures are stained blue, cytoplasm, muscle, erythrocytes and keratin are stained bright-red. Collagen is stained green or blue, depending on which variant of the technique is used.

Masson's Trichrome Stain (Luna 1968, Sheehan 1980)

Masson's Trichrome Stain (Luna 1968, Sheehan 1980) Results: ○ Muscle, RBC and keratin = ○ Nuclei = ○ Collagen and mucus =

red blue-black blue

GOMORI'S ONE-STEP TRICHROME STAIN Results ○ Muscle fibers = ○ Collagen = ○ Nuclei =

red green blue to green

Russell's modification of the Movat pentachrome stain The alkaline alcohol solution that follows converts the ______ _____ to __________ ________ _______, which is insoluble. Complete conversion is necessary because alcian blue will be decolorized during the remainder of the procedure._____ ____________ is used to stain the elastic fibers, which are then differentiated with ferric chloride.

alcian blue monastral fast blue Iron hematoxylin

Russell's modification of the Movat pentachrome stain are stained by alcian blue.

Acidic mucosubstances

__________________________ removes any residual iodine. ___________________ and __________________ are acid dyes that stain muscle, cell cytoplasm, collagen, and ground substance. ___________________________ differentiation removes the stain from the collagen and ground substance. The acetic acid removes the phosphotungstic acid, and collagen is then counterstained with alcoholic safran.

Sodium thiosulfate Crocein scarle acid fuchsin Phosphotungstic acid

Russell's modification of the Movat pentachrome stain Results: ○ Nuclei and elastic fibers = ○ Collagen = ○ Ground substance and mucin = ○ Fibrinoid, fibrin = ○ Muscle =

Black Yellow Blue Intense red Red

Several modifications of the original method have been developed. It is often used to differentiate acidophilic extracellular fibers from acidophilic cytoplasm. Basic ingredients are acid fuchsin, aniline blue, orange G and phosphotungstic acid.

Mallory's Aniline Blue Stain

Mallory's Aniline Blue Stain Results: ○ Nuclei, fibrin, muscle fibers = ○ Collagen = ○ Cartilage, bone, mucus varying shades of = ○ Blood, myelin = ○ Elastic fibers =

red blue blue yellow pale pink/yellow or unstained

This is Heidenhain's modification of Mallory's aniline blue stain, using azocarmine dye for counterstaining. It is a valuable stain showing minute details of connective tissue and of renal glomerular basement membrane as well.

Azocarmine Stain

Azocarmine Stain Results: ○ Amyloid connective tissues and mucous colloid - ○ Nuclei -

deep blue red

Elastic fibers are composed of the protein elastin and offer the greatest flexibility among the fiber types. Elastic fibers allow tissue to stretch and are located in the skin and walls of blood vessels. The presence of elastic tissue is recognized in acid-treated fresh preparations by a refractile appearance in contrast to the swollen glassy form of collagen.

Elastic Stain

Elastic Stain Common staining methods (5)

○ Weigert's Elastic Tissue stain ○ Taenzer-Unna Orcein method ○ Verhoeff 's stain ○ Gomori 's Aldehyde-Fuchsin stain ○ Krajian 's method

The tissue is placed in Weigert's stain, made up of basic fuchsin, resorcin and ferric chloride, differentiated with acid-alcohol, and counterstained with neutral red, H&E, or hematoxylin and van Gieson's stain.

Weigert's Resorcin-Fuchsin Elastic Tissue Stain

● Results: ○ Elastic fibers appear brown to purple or with blue black with methyl violet on a clear background ○ Other structures are colored depending on the counterstain used.

Histopath Proteins and Nucleic Acids Flashcards

(75 cards)