Specialized Prep Techniques

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Specialized Prep Techniques
Labels

• To identify the composition of the
  structure/organelle and/or phase of cycle.
• Identification of questionable structures.
• Follow cytochemical pathway through a time
  course.
• Localize genetic products, introduced compounds.

Cryo-fixation

• Structures are ephemeral or events are rapid.
• Structures are fixative sensitive.
• Removal of water changes topography/morphology

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Low Temperature Techniques Freeze Fracture
Sample is rapidly frozen, fractured and replica
is produced. Viewed with TEM. Cryo-Fixation
Can use a variety of methods to fix tissue prior
to substitution (removal of water). Cryo
-Microtomy Used in conjunction with
cryo-fixation to maintain frozen state of the
sample. Sections are kept frozen for viewing or
processed to dryness for conventional
viewing. Freeze Drying Easy and common
technique for SEM.
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Labeling Autoradiography Introducing an
isotope, then causing a reaction which deposits
an electron dense material at radioactive sites.
Lectin Lectins are small molecular weight
compounds that have variable affinities for
sugars. Usually bound directly to
label. Cytochemistry Chemical reactions that
require precipitation of colored reaction
product, heavy metal or electron dense material
for visualization. Immunolabeling Polyclonal
or monoclonal antibodies used to label
components. Usually used with fluorescent dye,
colloidal gold and sometimes silver
enhancement. In Situ Hybridization Using
nucleic acid hybridization techniques with
biotinylated nucleotides.
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Immune Responses
1. Humoral B lymphocytes produce antibodies
recognizing an antigen from foreign substance.
Antibodies are then secreted into blood stream.
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Immune Responses
1. Humoral B lymphocytes produce antibodies
recognizing an antigen from foreign substance.
Antibodies are then secreted into blood
stream. 2. Cell-mediated Mature T lymphocytes
- antigen responding, response control, and
response mediating cells
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Immunoglobins (Ig)
IgG
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Immunoglobins (Ig)
IgG
IgA
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Immunoglobins (Ig)
IgG
IgA
IgM
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Glossary
Antibody (anti-foreign body) is a protein
produced by a white cell (B lymphocyte).
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Glossary
Antibody (anti-foreign body) is a protein
produced by a white cell (B lymphocyte). Antigen
(antibody generating substance) is any agent,
such as a chemical or microorganism that is
recognized by the antibody. Not all antigens are
immunogens (e.g hapten).
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Glossary
Antibody (anti-foreign body) is a protein
produced by a white cell (B lymphocyte). Antigen
(antibody generating substance) is any agent,
such as a chemical or microorganism that is
recognized by the antibody. Not all antigens are
immunogens (e.g hapten). Immunogen Any
substance to which an animal responds by making
antibodies. All immunogens are antigens.
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Glossary
Antibody (anti-foreign body) is a protein
produced by a white cell (B lymphocyte). Antigen
(antibody generating substance) is any agent,
such as a chemical or microorganism that is
recognized by the antibody. Not all antigens are
immunogens (e.g hapten). Immunogen Any
substance to which an animal responds by making
antibodies. All immunogens are antigens. Antigen
binding site - relatively small region of an
antibody that binds to the antigen.
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Epitope (antigenic determinant) - is that part of
an antigen that is recognized by a single
antibody.
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Epitope (antigenic determinant) - is that part of
an antigen that is recognized by a single
antibody. Hapten - low molecular weight
compounds (such as plant hormones) that typically
do not elicit a spontaneous immune response but
can be recognized by antibodies. Typically
attached to an immunogen.
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Epitope (antigenic determinant) - is that part of
an antigen that is recognized by a single
antibody. Hapten - low molecular weight
compounds (such as plant hormones) that typically
do not elicit a spontaneous immune response but
can be recognized by antibodies. Typically
attached to an immunogen. Hybridoma - fusion
product between B cell and myeloma cell
(immortal cell).
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Epitope (antigenic determinant) - is that part of
an antigen that is recognized by a single
antibody. Hapten - low molecular weight
compounds (such as plant hormones) that typically
do not elicit a spontaneous immune response but
can be recognized by antibodies. Typically
attached to an immunogen. Hybridoma - fusion
product between B cell and myeloma cell
(immortal cell). HAT selection - culture media
that contains hypoxanthine, aminopterin and
thymadine. A selective media that only allows
hybridomas to grow.
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Terms used in Immunolabeling
Primary antibody An antibody that is specific
to the antigen of the sample to be localized.
Can be conjugated to a signal (gold, fluorochrome
or enzyme).
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Terms used in Immunolabeling
Primary antibody An antibody that is specific
to the antigen of the sample to be localized.
Can be conjugated to a signal (gold, fluorochrome
or enzyme). Secondary antibody An antibody
that recognizes a primary antibody. Usually
always is conjugated to signal.
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Terms used in Immunolabeling
Primary antibody An antibody that is specific
to the antigen of the sample to be localized.
Can be conjugated to a signal (gold, fluorochrome
or enzyme). Secondary antibody An antibody
that recognizes a primary antibody. Usually
always is conjugated to signal. Diluent
Physiologic buffer and non-specific protein (e.g.
albumin or non-fat milk) used in diluting the
antibodies. Sometimes detergent added to
decrease surface tension of sections.
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Block Physiologic buffer, high salt, and
non-specific protein. The protein adheres to any
sticky sites that might allow non-specific
binding of antibodies.
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Block Physiologic buffer, high salt, and
non-specific protein. The protein adheres to any
sticky sites that might allow non-specific
binding of antibodies. Etching treating resin
sections with HCl or sodium borohydride to reduce
steric hindrance or expose hidden antigenic
sites.
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Antibody Production
Polyclonal Antibodies are collected from sera of
exposed animal, - or - a combination of
monoclonal colonies is combined.
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Antibody Production
Polyclonal Antibodies are collected from sera of
exposed animal, - or - a combination of
monoclonal colonies is combined. Can be any
animal Rabbit, Goat, Horse, Rat, Sheep, etc
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Antibody Production
Polyclonal Antibodies are collected from sera of
exposed animal, - or - a combination of
monoclonal colonies is combined. Can be any
animal Rabbit, Goat, Horse, Rat, Sheep, etc
Suite of antibodies recognizing multiple
antigenic sites of injected biochemical.
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Monoclonal Individual B lymphocyte hybridoma
is cloned and cultured. Secreted antibodies are
collected from culture media.
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Monoclonal Individual B lymphocyte hybridoma
is cloned and cultured. Secreted antibodies are
collected from culture media. Typically BALBc
mice Sometimes Rat (ascites fluid).
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Monoclonal Individual B lymphocyte hybridoma
is cloned and cultured. Secreted antibodies are
collected from culture media. Typically BALBc
mice Sometimes Rat (ascites fluid).
Antibodies recognize one antigenic binding site
of the antigen.
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Determining which B cell to clone
ELISA or Immuno-fluorescence
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Generic light-level Immunolabeling Protocol
Fixation Formaldehyde (1-4 in physiological
buffer) Incubation in 10 Triton X-100 for 10
minutes (may require additional 10 minutes in
Methanol) Rinsing/rehydration in
buffer Immunolabel Mount in glycerol-based
medium or permanent mount. Usually contains
anti-fade (anti-quench) agent.
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Generic TEM Immunolabeling Protocol
Fixation 1 or less glutaraldehyde only may
include paraformaldehyde Dehydration Embedding
in methacrylate resin (e.g. LR White, Lowicryl,
or Quetol) Section Immunolabel Optional -
Post-stain
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Controls
Adsorption - primary antibody exposed to excess
of antigen to remove any labeling. Label without
antibody - no antibody, shows labeling not due to
reaction with label Omission of primary antibody
- the secondary or tertiary antibodies should not
recognize the tissue. Pre-immune sera -
collected from animals that have not produced
antibodies, or use normal serum from
non-immunized animals of same species
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Immunolabeling Sections
- Float grids on blocking solution - Incubate in
primary antibody - Wash thoroughly with
buffer to remove unbound antibody
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Incubate with secondary antibody or protein A/G
Wash again to remove unbound secondary Last wash
should be water.
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Double Labeling
Incubate sections with second primary antibody of
interest
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Incubate with secondary different fluorochrome,
or different size gold
Note steric hinderence at asterisk
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10 nm and 5 nm gold
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Microtubules labeled with anti-beta tubulin
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How are the antibodies coupled to the gold?The
proteins are attached to the gold particles
by a). Charge attraction (Lysine) b).
Hydrophobic attraction (Tryptophan) and c).
Sulfur binding (Cysteine and Methionine)
Strongest binding suggested to be the sulfur
"hinge" joining the the two Fc regions
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Using Protein A or Protein G instead of
secondary antibody
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Ferritin enhanced labeling
Silver enhanced
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Freeze fracture immunolabeled
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Freeze fracture immunolabeled
Negative stain Immunolabeled
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Etching to expose antigenic sites
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Arabidopsis root with anti-carbohydrate Abs
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Lectins The term lectin is a general term that
encompasses several families of proteins of
non-immune origin that bind glycoconjugates that
may or may not have a known function. Also
known as agglutinins since original discoveries
used agglutination of red blood cells
(recognition of surface sugars) as a
criteria. Many have secondary or even tertiary
affinities to other sugars - rigorous controls
required.
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1. A lectin molecule contains at least two
sugar-binding sites sugar-binding proteins with
a single site will not agglutinate or precipitate
structures that contain sugar residues, so are
not classified as lectins. 2. The specificity of
a lectin is usually defined by the
monosaccharides or oligosaccharides that are best
at inhibiting the agglutination or precipitation
the lectin causes. 3. Lectins occur in many
types of organisms they may be soluble or
membrane-bound they may be glycoproteins. 4.
Sugar-specific enzymes, transport proteins and
toxins may qualify as lectins if they have,
multiple-sugar binding sites.
IUPAC-IUB Joint Commission on Biochemical
Nomenclature (JCBN), and Nomenclature Commission
of IUB (NC-IUB)
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General Classes of Lectins
Animal 1) Galectins share galactose-specificity.
2) Ca-dependent (C-type) animal lectins form an
extremely large family, composed of members
having diverse structures and functions.
Selectins are a subfamily that have a specific
function in leukocyte adhesion to endothelial
cells through sialyl-LewisX recognition.
Collectins have a unique structure consisting of
a C-type lectin domain and a collagen-like
domain. They are supposed to be involved in
innate immunity.
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3) Invertebrates (such as snails - e.g. Helix
pomatia) are known to contain various lectins in
their body fluids, possibly as body-protection
factors. E.g. lectins from an echinoderm were
found to show hemolytic activity. 4)
Annexins have affinity to lipids with some having
binding activity to glycosaminoglycans.
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Plant 7) The legume lectin family consists of a
large number of members, such as Concanavalin A,
with variable saccharide specificities comparable
to C-type lectins. 8) Ricin was the first lectin
investigated in Russia more than 100 years ago
and is very toxic. It is now evident that ricin
has many other homologous members which differ in
either toxicity or sugar-binding specificities.
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Examples of commonly used Lectins
Ricinis communis (RcAI and RcAII) - Castor bean
a -D galactose, lactose (TOXIC!) Triticum
vulgaris (WGA) - Wheat germ N-acetylglucosamine
(trimers) (chitin) Helix pomatia (HpA) - Roman
snail N-acetyl galactosamine (cellulose) Canaval
ia ensiformis (Concanavalin A) - Jack bean a -D
mannose, a -D glucose Aplysia depilans (AGL) -
Aplysia gonad lectin Galacturonic acids Ulex
europaeus (UEA I) - Furze gorse seeds a -linked
fucose
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Lectin Protocol for EM
Osmium and Epon/Spurrs resin can be used. 1.
Sections collected on gold or nickel grids 2.
(Optional) Block and rinse thoroughly 3. Expose
lectin/gold conjugate to sections 4. Rinse
thoroughly 5. Post-stain
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Lectin Controls
Adsorption with primary affinity
substrate/sugar Adsorption with secondary and/or
tertiary affinity substrate/sugar Incubation
with unlabeled lectin Use of non-specific label
(e.g. BSA/gold complex) Incubation with
stabilized colloidal gold suspension
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Lectin labeling of a germinating fungal spore
Same regions (e.g. A) labeled with different
lectins to show distribution of wall
components top - chitin middle -
cellulose bottom - N-acetyl galactosamine
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Cytochemical Labeling
Identify carbohydrates, enzymes, proteins, etc
Intensity of catalytic activity can be
determined. Chemical reactions require gold
conjugate or electron opaque precipitate for
visualization. Can be pre-embedding or
post-embedding.
DAB reaction showing peroxidase in lysosomes
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Enzyme Labeling Reaction
Enzyme (recycled)
Exogenous substrate insoluble rxn
product Electron dense product (preci
pitant)
Trapping agent
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Exogenous substrate - reacts with endogenous
enzyme in tissue. The substance acted upon by
the enzyme is the substrate. (e.g. H2O2 with
peroxidase) Insoluble reaction product -
deposition and retention of the product
necessary for visualization. Trapping agent -
insoluble heavy metal salt (e.g. lead) or the
enzyme itself (e.g. HRP). Used to bind to and
contain or trap the reaction product and make it
directly visible. (e.g. DAB)
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Enzymes as organelle markers
Enzyme General Location Acid
Phosphatase Golgi, ER, Lysosomes Alkaline
phosphatase Plasma membrane Catalase Peroxisom
e (microbody) Cytochrome oxidase Mitochondria T
hiamine pyrophosphatase Golgi
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Tissues fixed in 2 glutaraldehyde in 0.1 M
sodium cacodylate buffer (pH 7.4) Washed
Incubated in 3,3'-diaminobenzadine
tetrahydrochlorine (DAB) and H2O2 Further
processed for transmission electron microscopy.
Ultrathin sections post-stained with uranyl
acetate and lead citrate.
Yang, Berin, Yu, Conrad and Perdue, J Clin
Invest, 2000, 106879-886
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Thyamine pyrophosphate in Golgi
Peroxidase in multivesicular body
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Requirements for Enzyme Reactions
Preservation of tissue structure and enzymatic
activity (formaldehyde and low
glutaraldyhyde) Maximization of reaction
conditions (temp, pH, optimal substrate and
trapping agent concentrations) Facilitation
of substrate penetration (typically limited to
100 mm - need to microtome prior to
embedding) Appropriate controls (lack of
substrate and/or trapping agent) Visualization
of reaction product (view sections prior to any
staining)
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Acid phosphatase in thick sections
200 kV
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Acid phosphatase in lysosomes
Acid phosphatase combined with immuno-localization
in lysosomes
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Miscellaneous Labeling Techniques
Modified Periodic Acid Schiff Reaction Aldehyde
groups are produced when periodic acid is used to
oxidize carbohydrates. These are then reacted
with alkaline silver solutions in which silver is
deposited at the reaction site Alkaline bismuth
stains certain polysaccharides incorporating the
PA-Schiff reaction.
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Tannic acid fix on human sperm
Actin filaments decorated with myosin
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In Situ hybridization as a localization technique
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In Situ hybridization labeling of HIV proteins