Transcript Histology - University of Mississippi Medical Center

Immunofluorescence
Immunostaining
1. Not a one size fits all approach
2. General guidelines that can be modified
depending on tissue, antigen and antibodies
available.
3. Optimization and luck are typically required.
Immunostaining General Steps
Overview
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Fix
Rinse (3x, 10 min)
Cryoprotect (if using frozen sections, 30 – 60% sucrose O/N, 4°C)
Imbed (paraffin, frozen, gelatin)
Section
Rinse (3x, 5 min)
Permeabilize
Non-specific block (1 hr, RT)
Primary Antibody (1 hr -72 hr, 37 °C, RT 4°C)
Rinse (3x, 5 min)
Secondary Antibody (1 hr, RT or 37°C)
Rinse (3x, 5 min)
Mounting media and coverslip
Fixatives
Options
1. Formaldehyde (CH2O)
2. Formalin, 10% (saturated, 37% formaldehyde) ***Often
has MeOH
3. Paraformaldehyde (4% PF); polymerized formaldehyde
4. Glutaraldehyde (high autofluorescence, ideal for EM)
5. Joy Juice (MeOH, acetone causes denaturation)
Time is important.
Fixatives cause crosslinking between proteins, leaving samples
in fixative more than 24-48 hrs can lead crosslinking
“blockade” of antigen.
Some antigen-antibody interactions are “fixative sensitive”, so
read antibody insert thoroughly.
To infuse or not to infuse
When should you infuse?
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Highly vascular
Large
Multiple organ collection
Approach
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Heparinize first
Anesthetize till respiration ceases
Open chest
Snip liver
Infuse with 10-60 ml PBS/saline, until fluid running out of liver is clear
(nose, paws, internal organs should blanch) via left ventricle.
Infuse with 10 – 60 ml fixative. Should get muscle contraction and
animal should get stiff like a board.
Excise samples and post-fix in fixative for 24-48 hours….Longer is not
better.
Permeabilization
Goal: allow antibodies access to intracellular
compartments
1. Detergents (TX-100, NP-40, Tween-20,
CHAPS, Saponin)
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Epitope dependent…don’t use if antigen is
“solubilized” by the detergent.
2. Solvents – minimize lipid barrier, DMSO
3. Fixative (PF, acetone, works with cultured
cells)
4. Optimization of time, duration and
concentration
Sectioning
Frozen
Advantages: quick, relatively easy, flexibility: works with most antigens
Disadvantages: can get cryo-damage, doesn’t preserve morphology as well as paraffin
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Cryoprotect with 30% sucrose (PBS) until sample sinks, usually O/N. Put in 60%
sucrose O/N if it doesn’t sink.
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Section thickness will vary with tissue …10 – 20 µm is goo general rule.
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Make sure chamber is cold (-20), blade is sharp and clean (wipe with EtOH if new to
remove oils)
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Getting good sections is an art and takes practice and constant
optimization/adjustments.
Paraffin
Advantages: preserves structure better, tissue samples keep longer
Disadvantages: usually requires “unmasking” techniques, requires more preparation time,
more likely to be incompatible with antigen-antibody interaction.
Gelatin
Use with vibratome
Can be desirable with brain sections
Blocking Solution
Purpose: Non-specific blocking of antigen sites that may
share some weak similarity to targe antigen. Prevents
antibody from binding off-target sites.
Choices:
commercial blocking reagents
NF milk
serum, ~5%
Never use serum from primary Ab host!!
Use serum from secondary Ab host, ~5% in PBS
Don’t mix goat and sheep antibodies in an
experiment!
If using a sheep primary Ab, don’t use goat serum to
block
Primary Antibody
Getting a good primary Ab can be challenging. Trial and Error.
Polycolonal
• Ab has multiple antigenic targets
• Rabbit, guinea pig, goat, sheep
• Non-renewable. Generating new ab means injecting new animals and
getting new serum which means the new antibody will not be identical to
the original.
• Commercial PC Abs will vary slightly from lot to lot, not usually a problem
from highly expressed antigens.
• Formats: serum, IgG purified, antigen purified (ideal)
Monoclonal: Ab has one target
mouse, some rabbit monoclonals available
• Renewable
• Format: ascites, purified
Multiple labeling
Ideal:
Multiple antibodies, each from different species
i.e. mouse, rabbit, sheep
If no other options, can use two antibodies from
the same host, but do “species conversion”
where you convert one mouse into a rabbit or
one rabbit into a mouse.
Secondary Antibodies
With fluorescence imaging, will need to use secondary Ab’s that
are conjugated to fluorophores that are compatible with
imaging device.
Confocal
488 (Cy2, Alexa 488)
561 (Alexa 555, Cy3)
633 (Alexa 633)
If using multiple labeling, make sure your secondary Abs have
been “pre-absorbed” against the other primary antibody
hosts or you will get cross-labeling.
Store in dark.
For MP, can use dyes excited at visible wavelengths, typically use
2x the visible wavelength to excite with MP.
Negative Controls
Negative Controls
• No primary, no secondary Ab (autofluorescence)
• No primary Ab, but include secondary Ab (secondary Ab
non-specific labeling)
• Always run one a no primary with every experiment!!!
• Antigen blockade (typically meaningless if Ab has been
affinity purified)
• Heterologous cell line expressing exogenous target
(trasfected cell line).
• siRNA knock-down
• IDEAL proof of Ab specificity: Knock-out/null animal.
Positive Controls
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Does Ab recognize target?
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Heterologous cell line expressing exogenous target
(trasfected cell line).
Does it label the type of cell I’m interested in
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VSMC- alpha smooth muscle actin, desmin
Endothelial cells – PECAM, CD31
Muscle fibers – myosin chain isoforms
Neurons – there are not a lot of good pan-neuronal
Abs, most are specific for specific populations; PG9.5,
neurofilament (heavy, intermediate, light)
Schwann cells – S100
Google it, go to literature
Signal low?
Try higher primary Ab concentration up to 1:25.
Try longer primary Ab incubation up to 72 hr.
Avoid fluorophore labeled primary antibodies (lose your
signal amplification with secondary Ab.
Signal Amplification with Tyramide conjugated fluorophore.
HRP conjugated secondary Ab, treat with H2O2, then
add tyramide conjugated fluorophore…get higher signal,
but some reduced resolution.
Look for signal directly at target, don’t be swayed by
“higher signal” in other tissues, different cell types have
different levels of background signal.
Other thoughts
Make your own antibody for commonly used sensitive targets. It’s expensive
upfront, but you’ll have the same Ab for many years and you’ll save time
repeatedly optimizing new Ab lots.
Write the receipt date on commercial Abs. They don’t last forever, even if stored
at -20.
Aliquot Abs into single use aliquots that are to stored at -20. Multiple freeze
thaws are bad.
Save all antibody information sheets and generate a catalog, write notes re: titer
and sensitivity.
Always read the antibody information sheet front to back.
Read thru catalogs, they usually have sections of fundamental information.
Always, always, always, run a negative control to account for “background” AF.
Don’t loose the tree thru the forest. Don’t expect your target to be the highest
fluorescing object in the tissue section. Background fluorescence in
different tissue varies and may be higher than in your expected target.
Compare apples to apples. This is why negative controls are important.
Just because an antibody works for western blotting, doesn’t mean it will work
for immunolabeling or vice versa.
Species specificity. Even though your antibody is supposed to be targeted to
mouse protein X, doesn’t guarantee it will or will not recognize that same
target in another species.