Basics of Immunohistochemistry

HHMI GATOR Workshop May 12, 2009 Lan Hoang-Minh

Immunohistochemistry (IHC)
y Overview of IHC y Care and use of microscopes y Magnification

y Immunohistochemistry uses antibodies to detect and

visualise antigens in cells and tissues y Extremely important research technique

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
y

Immunohistochemical Detection Methods

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

Immunohistochemistry Steps
Tissue sections Antigen retrieval Blocking endogenous enzymes Primary antibody Secondary antibody Chromogen Substrate Counterstain Mounting

Slide 3 of 23 Microscopy Observation

Immunofluorescence
y Fluorescence or confocal microscope y High structural resolution possible y Advanced image reconstruction (3D) and signal

quantification y Multiple labelling y Live cells

Direct Immunofluorescence Method

Easy application, only a few steps y Not very versatile, as primary antibodies need to be directly labelled with fluorophore
y

Fluorochrome-conjugated Primary Antibody Antigen expressing Cell

Indirect Immunofluorescence Method

y More steps involved y More versatile, only secondary

antibodies need to be labelled, different combinations of fluorophores possible in multiple labelling experiments

Fluorochrome-conjugated Secondary Antibody (anti species X)

Primary Antibody (from species X)

Antigen expressing Cell

Indirect Immunfluorescence Using Biotinylated secondary Antibody

Biotin (Vitamin B) binds with high affinity to Avidin good linker system y Very high sensitivity
y

Fluorochrome-conjugated Streptavidin Biotinylated Secondary Antibody (anti species X)

Primary Antibody (from species X)

Antigen expressing Cell

Fluorochromes have different emission wavelengths that produce different colors

Looking for new neurons in the adult brain

Use and care of microscopes Magnification

Major types of Light Microscopy

Brightfield : light focused on the specimen by condenser lens, then brought to eye via objective and ocular lenses; used with stains y Phase Contrast : uses condenser lens system to visualize differences of refractive index within cells and tissues; no stain needed y Fluorescence : uses light of a specific wavelength (e.g. UV), usually to visualize very specific stains that emit light at another specific wavelength y Confocal : uses scanning laser beam to make a series of sharp images on a photomultiplier tube, computers to record, then display these as a combined high resolution image
y

Brightfield

Darkfield

Fluorescence

Confocal

Parts of the Microscope

Using the Microscope

y Always observe the specimen or object using the LOWEST POWER objective

first. y Focus using the COARSE ADJUSTMENT KNOB to bring the object into focus. 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.

y Handling The Microscope y 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 not

in 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 damage the lenses.

Microscope Magnification
y Magnifying Power (MP) y Ocular lens y 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.

Field of view
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) must be calculated using the following equation:
y

FOV HP = Total magnification (TM) LP X FOV LP TM HP

Estimating the Size of the Specimen Under Observation

y 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.