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HHMI GATOR WorkshopMay 12, 2009
Lan Hoang-Minh
Basics of Immunohistochemistry
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Immunohistochemistry (IHC)y
Overview of IHCy Care and use of microscopes
y Magnification
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y Immunohistochemistry uses antibodies to detect and
visualise antigens in cells and tissues
y Extremely important research technique
y Antibodies can be raised against almost any type of
antigen (protein, carbohydrate, lipid...)
y Bind to antigen in a specific manner
y Can be detected in several ways
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Immunohistochemical DetectionMethods
y
Through fluorescent substances (fluorophores) Immunofluorescence technique
y Through enzymatic conversion (often by
horseradish peroxidase, HRP) of a soluble
substrate (chromogen) into a non-soluble and
colorful reaction product - Immunoenzyme
technique
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Immunohistochemistry Steps
Slide 3 of 23
Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Secondary antibody
Primary antibody
Microscopy Observation
Chromogen Substrate
Counterstain
Mounting
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y Fluorescence or confocal microscope
y High structural resolution possible
y Advanced image reconstruction (3D) and signal
quantification
y Multiple labellingy Live cells
Immunofluorescence
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Fluorochrome-conjugated
Primary Antibody
Antigen expressing Cell
Direct Immunofluorescence Methody Easy application, only a few steps
y Not very versatile, as primary antibodies need to be
directly labelled with fluorophore
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Antigen expressing Cell
Primary Antibody (from species X)
Fluorochrome-conjugated
Secondary Antibody
(anti species X)
Indirect Immunofluorescence Methody More steps involved
y More versatile, only secondary
antibodies need to be labelled,
different combinations of
fluorophores possible in
multiple labelling experiments
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Antigen expressing Cell
Primary Antibody (from species X)
BiotinylatedSecondary Antibody (anti species X)
Fluorochrome-conjugated
Streptavidin
Indirect Immunfluorescence UsingBiotinylated secondary Antibody
y Biotin (Vitamin B) binds
with high affinity toAvidin good linker system
y Very high sensitivity
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Fluorochromes have different emissionwavelengths that produce different colors
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Looking for new neuronsin the adult brain
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Use and care of microscopesMagnification
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Major types of Light Microscopy
y Brightfield : light focused on the specimen by condenser lens,then brought to eye via objective and ocular lenses; used with
stainsy Phase Contrast : uses condenser lens system to visualize
differences of refractive index within cells and tissues; no stainneeded
y Fluorescence : uses light of a specific wavelength (e.g. UV),usually to visualize very specific stains that emit light at anotherspecific wavelength
y Confocal : uses scanning laser beam to make a series of sharpimages on a photomultiplier tube, computers to record, thendisplay these as a combined high resolution image
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Brightfield Darkfield
Fluorescence Confocal
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Parts of the Microscope
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Using the Microscopey Always observe the specimen or object using the LOWEST POWER objective
first.
y Focus using the COARSE ADJUSTMENT KNOB to bring the object intofocus. Bring the object into sharp focus by using the fine adjustment knob.
y Focus, and then move to a higher power objective, if needed.
y Use only the FINE ADJUSTMENT KNOB when using the HIGHEST (longest)
POWER OBJECTIVE.
y Keep both eyes open to reduce eyestrain. Keep eye
slightly above the eyepiece to reduce eyelash interference.
y To find out the total magnification of the object,
multiply the power of the eyepiece lens (10X) by the
power of the objective.
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yHandling The Microscopey Always use two hands to move the microscope.
y Be gentle.
y Always have clean hands when handling your microscope.
y Storing The Microscope
y Dust is an enemy to microscope lenses; always keep the microscope covered
when not in use.
y Cleaning the Microscope
y
Dont let the microscope get too dirty always use the dust cover when notin use.
y To clean the eyepiece and objective lenses use a high quality lens paper.
First brush any visible dust from the lens, and then wipe the lens. Do not use
facial tissues, they are made from ground up wood fibers and could damagethe lenses.
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Microscope Magnificationy Magnifying Power (MP)
y
Ocular lensy Objective lens: 1 of 3 or 4 lenses that vary in MP. Low
power objective (LP), and high power objective (HP).
y MP microscope = MP ocular X MP objective
y E.g., if MP ocular = 10 and MP objective = 4, the MP of lens
combination = 40.
y If object is magnified 40 X, the image you see is 40 X larger
than the object would appear if viewed with the unaided eye
at a distance of ~ 25 cm.
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Field of viewy Field of view = maximum diameter visible through the lenses
of a microscope
y Place transparent metric ruler under the low power (LP)
objective. Focus microscope on the scale of the ruler. Measure the
diameter of the field of vision in millimeters. Record this number.
y The diameter of the field of view under high power (HP) mustbe calculated using the following equation:
FOV HP = Total magnification (TM) LP X FOV LP
TM HP
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Estimating the Size of the SpecimenUnder Observationy Convert your measurement to micrometers. Remember that 1 m =
0.001mm.y To estimate the size of an object seen with a microscope, first estimate
what fraction of the diameter of the field of vision that the
object occupies. Then multiply the diameter you calculated in
micrometers by that fraction.
y Eg., field of visions diameter is 400 m and the objects estimated
length is about 1/10 of that diameter, multiply the diameter by 1/10 to
find the objects length.
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