Immunostaining of cells and tissues

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Immunostaining of cells and tissues

• Megan Osler
• graduate student
• David Bader lab - Cell and Developmental Biology
• PRB 348 936-1971
• megan.emery_at_vanderbilt.edu

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Objectives

• Purpose of technique
• Scientific basis
• Methods of processing and detection
• Variations

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Purpose of Immunostaining

• To answer the question
• Where is my protein (antigen) located
• in a certain cell type or tissue?

Mouse gut Blue- goblet cells Red- actin in brush
border Green- stains nuclei
Endothelial cell Blue- tubulin Red-
F-actin Green- endosomes
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What tool can I use to identify my
protein? (hint what immunoreagent recognizes
circulating antigens in your body?)
Antibodies
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Antibodies specifically recognize a protein
epitope
Y

• Use an antibody raised to your protein and use it
  as a marker to search for the antigen in
  tissues/cells.

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Generating (monoclonal) antibodies
Starting product Some or all of your protein
N
C
(you may be interested in this region)
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How do you go from to ???
Y
Y
Y
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Method (most common)

• Indirect Immunofluorescence
• Double antibody technique for signal amplification

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Method (most common)

• Indirect Immunofluorescence
• Double antibody technique for signal amplification

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How does the 2? recognize the 1??
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2? binds the Fc portion of 1?
How does the 2? recognize the 1??
Y
Y
Fc
Very important You must match the species!
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Method (most common)

• Indirect Immunofluorescence
• Multiple labeling

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Y
Y
Y
Y
Y
Y
Y
Y
Y
Why double label?
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Procedure overview

• Prepare sample
• Fixation/Permeabilization
• Blocking
• Primary antibody incubation
• Secondary antibody incubation
• Prepare for viewing
• variations

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Cell/Tissue preparation

• Cells
• grow on cover slips or
• in chamber slides

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Fixation Purpose to preserve the cells/tissue
and to immobilize the antigen
GOOD
fix

• Common fixatives
• PFA (1-4)
• Alcohol (MeOH or EtOH)
• Histochoice (commercial)
• How do you choose which fix to use?
• Antibody dependent

live cell
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Permeabilization Purpose to punch holes in
the cell membrane so antibodies can diffuse in to
bind the target protein
Y
Y
Y
Perm.

• Permeabilizing agents (cells only)
• Alcohols (also a fixative)
• Detergents (Triton-X, Tween)

Antibodies can bind intracellular proteins
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BlockingPurpose to eliminate non-specific
binding of antibodies
Reagent (most common) 2 Bovine Serum Albumin
(BSA)
Y
Y
Sticky proteins bind the BSA, so your antibody
will only bind YOUR PROTEIN
BSA
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Antibody incubationPurpose bind your protein,
amplify signal
Cells blocked with BSA
Wash with PBS in between steps and Perform in a
humid chamber to prevent drying out the slide
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Prepare for viewing Mount slide

• Purpose protect slide add anti-fade reagent
• Place coverslip on slides with mounting reagent
  (Polyaquamount)
• Keep in the dark until viewing

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Important controls

• Pre-immune serum/2? Ab control
• Why? To determine that labeling is not background
  from the 2? Ab
• Ab that you KNOW will work (actin)
• Why? To verify that your procedure worked
• Dilution series
• Why? To optimize the concentration of the
  antibodies for the best possible signal

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Immunofluorescence

• Pros
• Easy
• Fast- 1 day
• High contrast
• Confocal
• Attractive images

• Cons
• Fluorescent microscope
• Bleaching
• Not permanent

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One more thing

• Not every fluorescent signal originates from an
  antibody.
• There are STAINS also.
• Uses? Cytotoxicity, apoptosis

DAPI- nuclear stain
MitoTracker- mitochondria
Phalloidin- F-actin
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Tricks for success

• PAP pen
• Nail polish
• VWR marker
• Q-tips

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Potential Issues
Signal Intensity antibody avidity to protein 2?
Ab binding to 1? Ab
Y
Y

• Epitope masking

Y
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Variations

• Immunohistochemistry (enzyme)
• AP, HRP, NBT-BCIP, etc. detection systems
• Permanent, bright field microscope
• Takes longer, product may diffuse

Anti-Trk B receptor
Incubation with diaminobenzidine (DAB, 7 min,
RT), in the presence of H2O2. Polymerization of
the benzidine results in a permanent brown
precipitate proportional to the amount of antigen
present.
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Variations

• Immunogold EM (heavy metal)
• Permanent, detect Ag at ultrastructural level
• Difficult, tedious to optimize

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Fluorescence in situ hybridization
(FISH)technique to directly identify a specific
region of DNA or RNA in a cell
Ex An enzyme-linked RNA probe binds an RNA -
antibody recognizes enzyme - another
fluor-conjugated antibody recognizes first Ab
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Tyramide Signal Amplification (TSA)
HRP
Fluorophore-tyramide
Uses HRP (enzyme) to catalyze the deposition of a
fluorophore-labeled tyramide amplification reagent
Tyramide Signal Amplification Method in
Multiple-Label Immunofluorescence Confocal
Microscopy Guoji Wang et al. Methods. Volume 18,
Issue 4 , August 1999, Pages 459-464
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Problem 1 You have started a new rotation in a
lab that has just discovered a novel protein
called LEK. It is your rotation project to
characterize the distribution of this protein in
a skeletal myoblast cell line, C2C12. You have a
primary monoclonal antibody mouse anti-LEK You
have a wide range of secondary antibodies to use-
many species and many colors. Knowing nothing
about the protein, you used a general protocol,
as follows Fix in 2 PFA for 20 minutes Block
with BSA for 1h at RT Incubate cells with your
primary antibody 1 hr at RT Incubate with a
secondary antibody that is conjugated with a
GREEN fluor along with DAPI, which stains the
nucleus blue 1hr at RT Mount your slide and
view This is what you see under the
microscope. Oops- something isnt right. You
dont see any green label. You repeat it
again, altering one step, and this is what you
see. Green staining INSIDE the cell. What are
2 changes that you could have made in your
procedure to correct your mistake and observe
staining of your protein. (hint one is before
the blocking step and one is after)
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