IHC and Using Microscopes - Lan Hoang-Minh

7/29/2019 IHC and Using Microscopes - Lan Hoang-Minh

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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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Primary Antibody (from species X)


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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Primary Antibody (from species X)

BiotinylatedSecondary Antibody (anti species X)


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.

IHC and Using Microscopes - Lan Hoang-Minh

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