STAINING PROTEINS AND

NUCLEIC ACIDS
BARTE ꟾ NGAMOY ꟾ OMAWENG
 Simple
PROTEIN HYDROLYSIS AMINO ACIDS & OCCASSIONAL CARBOHYDRATE COMPOUNDS

 Conjugated
COMPLEX PROTEIN PROTEIN + NON-PROTEIN

 Derived
PHYSICAL
SIMPLE/CONJUGATED PROTEIN or DERIVED PROTEIN
CHEMICAL
 Fibrous
forms muscle fibers, tendons, connective tissues, bone
 Globular
more soluble than other classes; transports, catalyzes,
regulates
 Membrane
relays signals within cells; transports and allows
interactions
 DEOXYRIBONUCLEIC ACID (DNA)
5-carbon deoxyribose
nucleus of the cell
adenine & thymidine; guanine & cytosine

 RIBONUCLEIC ACID (RNA)

cytoplasm > nucleus (nucleolus)
ribose sugar
adenine & uracil; guanine & cytosine
 pH: 4-8
 Acetic acid Methylene Sharpens
1-2% aq. Solutions of neutral red Blue Nuclear
Borax Staining
 Acidophilic Dyes Mitochondria & Collagen (Anionic)
 Mallory Staining Technique – aniline blue, acid fuchsin, orange g
 Basophilic Dyes Basic Substances
 Hematoxylin – Derived From Hematein
 used with mordant (aluminum salt) forming hemalum
 stains blue to purple
 substituted by:
ethylene blue, toluidine blue, & thionine (imposes metachromasia)
carmine, basic fuchsin, and azure ii
 Eosin Y – Used As Counterstain During Nuclear Staining
 stains red, pink, or orange
  Demonstrates presence of amino acid molecules
 Based upon the identification of specific linkages or group within
the amino acid molecule
 Neutal-buffered Formol Saline – most common fixative for amino
acid histochemistry
 Glycol Methacrylate – plastic embedding medium commonly used in
histopathology

 0.3-2 µm aniline black & coomasie brilliant blue R 250 microscopy

 Fast Green – acid dye that stains basic protamines and
histones
 Trichloroacetic Acid – removes nucleic acid in basic
protamines and histones

 Peracetic Acid – oxidizes cystine & cysteine forming strong

cysteic acid (stained blue-green)
 Sakaguchi’s Test – used for arginine
 Proteins that are heavily glycosylated
 Occur in the connective tissue and major component of
extracellular matrix
 Mucopolysaccharidoses – inability to break down proteoglycans

 Utilizes properties of both pas and alcian blue methods

 Technique is to stain all acidic mucins with alcian blue
blocking the remaining pas positive. Neutral mucins will
then be stained and demonstrated in contrasting manner
Demonstrates both DNA and RNA

 PEARSE 1968; CARSON 1983

 Used to identify chromosomal material or DNA in cells
 uses 1 M HCl @ 60⁰C to hydrolyze purine-deoxyribose bond
exposing aldehydes, then stained by Schiff’s reagent demonstrating
DNA
 results in purple staining
 DNA - specific
 Bancroft & Cook 1994
 uses basic dyes to produce differential staining reaction for
DNA & RNA
 Methyl Green – DNA
 Pyronin - RNA
 DNA results in green color; RNA results in pinkish to red color
 utilized to detect the presence of plasma cells and
lyphocytes
 uses fluorochromes to produce light or visible radiation energy when
excited by light of shorter wavelength
 includes need for special light source
 Fluorochrome tracers:
1. Fluorescein – most widely used
2. Rhodamine – commonly used in two-color techniues
3. Acridine orange – demonstrate both DNA & RNA in fresh or fixed tissues
4. Acriflavine – alternative to basic fuchsin in Schiff’s reagent
 Immunohistochemical Staining – most common applied protein
immunostaining technique
 Immuno-electron Microscopy – allows detection of specific
proteins in ultra-thin tissue sections
 technically challenging, expensive, require rigorous
optimization of tissue fixation and processing methods
 Formalin-fixed, Paraffin-embedded (FFPE) Tissues
can demonstrate nucleic acids even decades after fixation

 Rapid Fresh Frozen Tissues using Liquid Nitrogen

for tissue antigens that cannot survive even moderate aldehyde fixation
poor morphology, poor resolution at higher magnification, difficulty in
cutting, need for frozen storage
 Glutaraldehyde (2.5% Solution In Phosphate-buffered Saline)
most commonly used fixative
 Phosphotungstic Acid
used as protein precipitant for positive staining of whole muscle and
collagen fibrils
 Aldehyde fixation Phosphotungstic acid intense electron-
opaque stain
 Intensity of stain reflects the concentration of protein
 Technique widely used to separate biological macromolecules
 Stains that allow visualization of proteins:
 Coomassie brilliant blue R-250
most popular; recommended for mass spectrometry and protein
identification
 Ethidium bromide
sensitive for DNA; a potent mutagen
 Silver stain
offers highest sensitivity; time-consuming, complex
 Fluorescent stain
ideal but expensive; requires a CCD (charged-coupled device)
camera or fluorescent scanner for gel imaging
 Definitive and most sensitive technique for identifying DNA
labeled complementary specific DNA, RNA,
DNA, RNA, or modified or nucleic acid Quantitate
nucleic acid strand sequence
 FISH (Fluorescent In Situ Hybridization)
 can visualize multiple targets simultaneously and co-localized multiple gene
expression patterns with a single specimen using spectrally distinct fluorophere
 CISH (Chromogenic In Situ Hybridization)
 reveals genetic information in the context of tissue morphology using methods that
aare aalready available in histology laboratory
 3 major steps involved in PCR
 1. Denaturation
 heating of DNA @ 95⁰C creating 2 separate single stranded DNA

 2. Primer Annealing
 cooling of mixture at medium temperature (54⁰C) to allow binding of primers
to complementary single stranded DNA template.
3. Extension
 heating @ 72⁰C for the attachment of DNA polymerase and start copying the
double stranded DNA molecule
 Reverse Transcription PCR (RT-PCR)
 most sensitive technique for mRNA detection and quantitation
 use of reverse transcriptase to form complementary DNA from the
desired RNA template

 In Situ Hybridization
 detects minute quantities of rare or single-copy number nucleic
acid sequences in frozen or paraffin-embedded cells or tissue sections