HUZAIFA UMARDEPARTMENT OF BIOENGINEERING

STUDENT NO: 20142894CYPRUS INTERNATIONAL UNIVERSITY

15TH

December, 2014 1

Outlines

Introduction

General Application of Biomaterials

Cellular Measurements in Proteomics

Protein Immunostaining

Immunohistochemistry/Immunocytochemistry

Application of IHC

Flow Cytometry

Application of Immunostaining

Conclusion

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Definition: Biomaterial

Any material of natural or of syntheticorigin that comes in contact with tissue,blood or biological fluids, and intendedfor use in prosthetic, diagnostic,therapeutic or storage applicationswithout adversely affecting the livingorganism and its components.

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What is a biomaterial?

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Therapeutic Devices

in Orthopedics

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Applications in Dentistry

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Catheters

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In Ophthalmology

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In Nephrology

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In Drug Delivery

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Future Applications- Tissue

Replacement

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In Diagnosis

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Biomaterials Represent a

Broad Range of Products

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General Applications of

Biomaterials

Storage of fluids, tissues, and other

biological products

Diagnosis

Monitoring

Therapy

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Biomaterials can be derived either fromnature or synthesized in the laboratory usinga variety of chemical approaches utilizingmetallic components, polymers, ceramics orcomposite materials.

They are often used and/or adapted for amedical application, and thus compriseswhole or part of a living structure orbiomedical device which performs,augments, or replaces a natural function.

Biomaterials are also used every day indental applications, surgery, and drugdelivery.

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Cellular Measurements in Proteomics

Common methods of protein detection are:-

Immunohistology/Immunocytology

Flow Cytometry

Western Blotting

ELISA

Immunoprecipatation

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Immunohistochemisty

IHC is a technique that uses antibodies (matchingmolecules) that can seek, identify and attachthemselves to these markers on cells. The antibodiesthemselves can be seen under microscope, whichhelps the technician to make a preciseidentification.

For example, several diseases or disease sub-typesmay look alike or appear to have similar size cellsunder a microscope but have different behavioursand necessary treatments. The best way todifferentiate them is to detect specific molecules onthese cells that act as markers.

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Protein immunostaining

is a general term in biochemistry that applies toany use of antibody-based method to detect aspecific protein in a sample.

The term immunostaining was originally used torefer to the immunohistochemical staining oftissue sections, as first described by Albert Coonsin 1941. Now however, immunostainingencompasses a broad range of techniques used inhistology, cell biology, and molecular biology thatutilize antibody-based staining method

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Immunochemistry - what’s

good about it?

Antibodies bind to antigen in specific manner

Gives a spatial location

Can be used to locate particular cells and proteins

Can be used to identify cellular events- e.g. apoptosis

And it can also be use in cancer diagnosis (Lymphomas).

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Identify replicating cells

Examine cytoskeletal structure

Identify activation states

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Locate cells that are signaling

Locate apoptotic cells

Identify different types of cells in a tissue

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Immunohistochemistry Principle

IHC is the process whereby antibodies are usedto detect proteins (antigens) in cells within atissue section (e.g. Liver).

Major components in complete IHC experiments:

Primary antibody binds to specific antigen;

The antibody-antigen complex is formed byincubation with a secondary, enzyme-conjugated, antibody;

With presence of substrate and chromagen, theenzyme catalyzes to generate coloured depositsat the sites of antibody-antigen binding. 23

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IHC Processes

Sample Preparation

Fixation(Paraformaldehyde, Alcohol)

Embedding

Sectioning

Antigen retrieval

IHC Protocol

Blocking (Peroxide and power block)

Incubation with Primary Antibody

Super Enhancer

Incubation with Secondary Antibody

Adding enzyme substrate

Cover slip and observation using microscope

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Goat anti-actin

Flourochrome (488)

conjugated

streptavidin

Biotinylated donkey anti-

goat

Enzyme linkage indirect method

Enzymatic detection methods

Brightfield microscope sufficient for analysis of specimens

Suitable for tissue analysis at low magnification

Resolution of subcellular structures not as good as with fluorescence methods, but can be combined with electron microscopy

Unimited shelf life of labelled specimens

Substrate reagents often toxic/carcinogenic

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Flow cytometer is a technology that allows a single cell to be measured

for a variety of characterestics, determined by looking at how it flow in

liquid. Instruments used for this can gather information about cells by

measuring visible and flourescent light emissions, allowing cell sorting

based on physical, biochemical and antigenic traits.

It can be used for the direct analysis of cells expressing one or more

specific proteins, Cells are immunostained in solution using method

similar to the one used for immunofluorescence, and then analyzed by

flow cytometry.

Flow cytometry has several advantages over IHC including: the ability

to define distinct cell populations are defined by their size and

granularity; the capacity to get out dead cells; improved sensitivity; and

multi-colour analysis to measure several antigens simultenously.

However, flow cytometry can be less effective at detecting extremely

rare cell populations, and there is a loss of architectural relationships in

the absence of a tissue section.

Flow Cytometry

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Front view of a desktop flow cytometer - the Becton-Dickinson

Fluorescence activated cell sorter (FACSCalibur)29

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Conclusion

IHC has strengths and weaknesses, sothink about your planned assay beforeacquiring tissue

Good block, appropriately fixed andsectioned can give you great data

Bad block, inappropriately fixed andsectioned, can give you misleading dataand waste money

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Questions?

Comments/Observations

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