STAINING OF PROTEINS AND NUCLEIC ACIDS

Protein 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.

Based on chemical composition:

Simple proteins
 On hydrolysis they yield only the amino acids and occasional small carbohydrate
compounds.
Conjugated proteins
 These are simple proteins combined with some non-protein material in the body to form
complex proteins.
Derived proteins
 These are proteins derived from simple or conjugated proteins by physical or chemical
means.

Three types of proteins based on physical configuration:

 Fibrous Proteins
 Form muscle fiber, tendons, connective tissue and bone.
 Fibrous proteins can be demonstrated by selective staining with small or large molecule
dyes (trichrome method), and silver impregnation (reticulin method), and specific dye-
protein interactions (e.g., Congo red stain for amyloid).
 Globular proteins
 More water soluble than the other classes of proteins and they have several functions
including transporting, catalyzing, and regulating.
 Globular proteins are found in blood and tissue fluids in amorphous globular form with
very thin or non-existent membranes.
 Membrane Proteins
Play several roles including relaying signals within cells, allowing cells to interact, and
transporting molecules. Membrane proteins often serve as receptors or provide channels
for polar or charged molecules to pass through the cell membrane.

NUCLEIC ACIDS

Nucleic acids are usually combined with basic proteins to form nucleoproteins.
Consist of alternate sugar and phosphate groups, with a nitrogenous base attached to each sugar
group.
Two major nucleic acids
  Deoxyribonucleic acid (DNA) contains a 5-carbon sugar deoxyribose, and is mainly
found in the nucleus of the cell. The four nitrogenous bases of DNA are purines (adenine
and guanine) and pyrimidines (thymine and cytosine).
 Ribonucleic acid (RNA) is found in the cytoplasm, and to a lesser extent in the nucleus,
particularly in the nucleolus. It contains ribose sugar with attached nitrogenous bases of
purines (adenine and guanine) and pyrimidines (uracil and cytosine).

Hematoxylin & Eosin Stain (H & E)

 Staining method involves application of hemalum, a complex formed from aluminum ions and
hematin (an oxidation product of hematoxylin).
 Hemalum colors nuclei of cells (and a few other objects, such as keratohyalin granules and
calcified material) blue
 The nuclear staining is followed by counterstaining with an aqueous or alcoholic solution of eosin
Y, which colors eosinophilic structures in various shades of red, pink and orange.
 Cytoplasm is eosinophilic. Red blood cells are stained intensely red.

Histochemical Identification of Proteins

Histochemical methods are used to demonstrate the presence of amino acid molecules rather than
whole protein molecules.
Neutral buffered formol saline is the most commonly used fixative for amino acid histochemistry.
The plastic embedding medium (glycol methacrylate) is commonly used in histology and
pathology because some of the artifacts (shrinkage and distortion) caused by hot paraffin can be
largely avoided.

Alkaline Fast-Green Method for Basic Proteins (especially protamines and histones)

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.

Peracetic Acid-Alcian Blue for Cystine and Cysteine

Peracetic Acid oxidizes cystine and cysteine, forming strong cysteic acid which is stained
blue-green by a basic dye. Sakaguchi’s test for arginine uses NaOH, sodium hypochlorite
(Milton's reagent) and pyridine chloroform, producing orange-red color on objects
containing arginine.
Proteoglycans

Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit
consists of a "core protein" with one or more covalently attached glycosaminoglycan
chain(s).
Occur in the connective tissue and are a major component of the extracellular matrix.

Alcian Blue-PAS Staining for Proteoglycans

This is a combined method utilizing the properties of both the PAS and Alcian blue
methods to demonstrate the full complement of tissue proteoglycans.

STAINING OF NUCLEIC ACIDS

 Demonstration of nucleic acids depends upon either reaction of the dyes with the phosphate
groups, or production of aldehydes from the sugar (deoxyribose).
 Feulgen technique - demonstrates sugar
 Methyl green pyronin technique - demonstrates phosphate
 Acridine orange (by fluorescent method)
 Gallocyanin-chrome alum method demonstrates both DNA and RNA

*This last staining method does not separate the two nucleic acids since it stains both DNA and RNA
blue, and suitable extraction technique must be used.*

Feulgen Staining for Nuclear DNA (Pearse 1968; Carson 1983)

Feulgen stain is a staining technique used to identify chromosomal material or DNA in

cell specimens.
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.

Methyl Green-Pyronin method for RNA and DNA (from Bancroft & Cook 1994)

Methyl 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.
Methyl green-pyronin stain is also utilized to detect the presence of plasma cells and
lymphocytes.

Fluorescent Staining for DNA and RNA

  Fluorochromes are fluorescent dyes which emit light or visible radiation energy when excited by
light of shorter wave length, either visible or ultraviolet.
 Fluorescein is still the most widely used fluorochrome, because of its wide
absorption spectrum and blue light range. Its characteristic apple green emission
is rarely seen as "auto fluorescence" in mammalian tissue, which is often blue in
color.
 Rhodamine conjugates absorb maximally in green light, exhibiting an orange-red
emission, and are commonly used in two-color techniques.
 Acridine Orange is the most commonly used fluorochrome to demonstrate DNA
and RNA in fresh or fixed tissues, combining with nucleic acids in cells by salt
linkages and cohesion.
 Acriflavine, as a 0.01% alcoholic solution, can be used as an alternative to basic
fuchsin in Schiff's reagent, for the Feulgen technique of acid hydrolysis.

Immunohistochemistry

 Immunohistochemical staining of tissue sections is perhaps the most commonly applied protein
immunostaining technique.

Antigen Retrieval

 Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at room

temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after
fixation.

Electron Microscopy

 The most commonly used fixative is glutaraldehyde, usually as a 2.5% solution in phosphate
buffered saline.
 The main action of these aldehyde fixatives is to cross-link amino groups in proteins through the
formation of methylene bridges (-CH2-), in the case of formaldehyde, or by a C5H10 cross-links
in the case of glutaraldehyde.

Polyacrylamide Gel Electrophoresis

 Polyacrylamide gel electrophoresis (PAGE) is a technique that is widely used to separate

biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic
mobility.
Stains that allow visualization of the protein pattern in the gel:

Coomassie Stains
o The most popular anionic protein dye, Coomassie Brilliant Blue, stains almost all
proteins. Coomassie Brilliant Blue: R-250 (R for reddish) offers shorter staining times
than G-250 (G for greenish). Coomassie dyes are also the favorite stains for mass
spectrometry and protein identification.
Ethylene Bromide
o Ethidium bromide is a sensitive, easy stain for DNA.
Silver Stains
o Silver stains offer the highest sensitivity, although protocols are often time-consuming,
complex, and do not offer sufficient reproducibility for quantitative analysis.
Fluorescent Stains
o These stains are ideal for protein study but are more expensive than Coomassie or silver
stains and require either a CCD (charge-coupled device) camera or fluorescence scanner
for gel imaging.

IN-SITU HYBRIDIZATION

The technique uses a labeled complementary DNA, RNA or modified nucleic acids strand (or
probe) to localize a specific DNA, RNA, nucleic acid sequence or gene expression within a cell,
in contrast with immunohistochemistry, which usually localizes proteins in tissue sections. After
hybridizing a labeled complementary DNA or, a complementary RNA (riboprobe) to the target
sequence at elevated temperature, the excess probe is washed away (after prior hydrolysis using
RNase in the case of unhybridized, excess RNA probe), and the probe that was labeled with either
radio-, fluorescent- or antigen-labeled bases (e.g., digoxigenin) is localized and quantified in the
tissue either by autoradiography, fluorescence microscopy, or immunohistochemistry,
respectively.

Polymerase Chain Reaction (PCR)

In-situ hybridization techniques are now also supplemented by the polymerase chain reaction
(PCR) method whereby a single copy or a few copies of a piece of DNA can be amplified across
several orders of magnitude, generating thousands to millions of copies of a particular DNA
sequence. In-situ PCR potentially provides a means of detecting single copies of nucleic acid
sequences in cellular preparations.
The method relies on thermal cycling, through cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic replication of the DNA.

Reverse transcription polymerase chain reaction (RT-PCR)

 One of many variants of polymerase chain reaction (PCR), is the most sensitive technique for
mRNA detection and quantitation currently available.

In Situ PCR

In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that detects minute
quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded
cells or tissue sections for the localization of those sequences within the cells.