Immunohistochemistry

Immunohistochemistry & Competitive Inhibition

The plasma membrane of a cell is composed of a lipid bilayer, proteins and carbohydrates. Plasma membrane proteins can be embedded across the membrane (transmembrane proteins) or anchored to the membrane on the extracellular or intracellular surface (peripheral proteins). Transmembrane proteins are often bound to oligosaccharide chains (sugar chains) on the extracellular side of the plasma membrane (glycoproteins). Many of these glycoproteins are used as means for cell communication/recognition and are referred to as cell-surface receptors.

A good example of how we can use antibodies to identify membrane proteins or carbohydrates (antigens) when we test a person for their blood type.

The red blood cells (RBCs/erythrocytes) can contain the glycoproteins A and/or B or neither that make up the A, B, O system used to determine blood type.

An antigen is defined as ANYTHING that an ANTIBODY will bind to. When an antibody binds to its antigen, the cells clump together (agglutinate).

If agglutination occurs with antibodies against glycoprotein A BUT NOT with antibodies against glycoprotein B, then the blood type is type A.

If agglutination occurs with antibodies against glycoprotein B BUT NOT with antibodies against glycoprotein A, then the blood type is type B.

If agglutination occurs with BOTH glycoprotein A and B (type AB blood) then the blood type is type AB.

If agglutination DOES NOT occur with EITHER glycoprotein A or B then the blood type is type O.

The agglutination (clumping) process is helped by lectins. Concanavalin A (ConA) is a lectin (carbohydrate-binding protein) Lectins are proteins that bind to the oligosaccharide chains on glycoproteins on the plasma membrane of RBCs.

Immunohistochemistry is a technique that physiologists can use to determine if certain molecules are present in the sample.
Antibodies are made that will bind to certain amino acid sequences. Glycolipids and glycoproteins have specific carbohydrate chains that help create receptors. Immunohistochemistry allows us to use lectins (a protein molecule that will bind to specific carbohydrates and tends to be isolated from plants) to determine the carbohydrate make-up of the cell surface receptors of a cell.

LECTIN

The agglutination (clumping) process is helped by lectins. Concanavalin A (ConA) is a lectin (carbohydrate-binding protein)
Lectins are proteins that bind to the oligosaccharide chains on glycoproteins on the plasma membrane of RBCs.

In this lab you will determine if cheek epithelial cells express cell surface receptors that bind Concanavalin A (Con A). By adding various free carbohydrates (monosaccharides), we can determine if these free carbohydrates compete with the receptors on the cell surface.

If the free carbohydrates are competitive inhibitors, then lectin (Con A) will bind to both the free carbohydrate and the receptor and we can conclude that the cell surface receptor contains the same structure as the free carbohydrate. If the free carbohydrates are not competitive inhibitors, then the lectin will only bind to the cell surface receptor and we can conclude that the receptor does not contain the same structure as the free carbohydrate. You will use two ways to visualize the lectin (Con A) binding to the cell surface receptors: 1) colorimetric (color change) and 2) hemagglutination (seeing red blood cells clump/stick together). Your objective is to identify the type of sugar chains found on Con A cell surface receptors that allow Con A (lectin) to bind.

The Chemical Structure of Mannose

Lectins are carbohydrate-binding proteins, macromolecules that are highly specific for sugar moieties of other molecules. They are also known as phytohemagglutinins. Lectins perform recognition on the cellular and molecular level and play .... Lectins are similar to antibodies in their ability to agglutinate red blood cells.

A type of Lectin called concanavalin A, have been used widely as model systems to understand the molecular basis of how proteins recognize carbohydrates, because they are relatively easy to obtain and have a wide variety of sugar specificities. The many crystal structures of legume lectins have led to a detailed insight of the atomic interactions between carbohydrates and proteins.

Concanavalin A (Con A) is a plant lectin that is purified from jack beans.

Con A binds to the mannose residues of various glycoproteins

Con A DOES NOT bind to galactose residues of various glycoproteins

Lab exercises:

LAB A. Antigen-Antibody interaction simulation
Equipment:

● Textbook/resources
● Immunohistochemistry demo kits

Protocol:

1. Instructor will walk you through a demo for cheek cell experiment (Lab Exercise B).
2. Run the demo for the red blood cell experiment (Lab Exercise C).
3. Clean-up: Return all the Immunohistochemistry demo kits; make sure all pieces are inside the bag.

After this demo you should be able to explain the following concepts: specificity, competitive inhibition, antibody, antigen, lectin, binding site, ligand, substrates, and hemagglutination.

B. Immunohistochemistry-Cheek Cells
Equipment

● Wax pen
● Microscope
● 2 Slides
● 4 Cover slips
● Tooth picks
● Ethanol
● Microliter pipets
● Transfer pipets
● Small beakers
● Microcentrifuge tubes
● Con-A Buffer (pH 6.8)
● Con-A Peroxidase (Con-A peroxidase + Con-A buffer)
● 1M Galactose (Galactose + Con-A buffer)
● 1M Mannose (mannose + Con-A buffer)
● Peroxidase Substrate Solution (hydrogen peroxide + chloronapthol + Tris buffer)

Protocol:

1. You will place two samples on each slide.
2. Label each side of the slide either: #1, #2, #3, or #4.
3. Using a wax pen, draw a circle next to each label. This is where you will create your cheek cell smear.
4. Rinse your mouth with DI water to remove bacteria, food, mucus, etc. Take a clean toothpick and gently scrape the inside of your cheek to remove simple squamous epithelial cells.
5. Smear the cells on the slide labeled #1 and allow it to air dry.
6. Repeat step 7 for slide #2, #3, and #4.
7. Fix (makes cells permeable to macromolecules and causes them to adhere to the slide) the cheek cells onto the slide by using 1 drop of ethanol on the cheek cells and allowing the slide to air dry.
8. Repeat step 7.
9. Hold the slide at an angle and slowly rinse each slide with 1mL of Con-A Buffer. Remove excess buffer by taping the slide on its side.
10. Repeat step 9.
11. Label 4 microcentrifuge tubes: #1, #2, #3, and #4.
12. Fill each microcentrifuge tube accordingly and mix well.
13. Place the slides on the bench top and dispense accordingly.
- a. Dispense 15μL of solution #1 onto slide #1.
- b. Dispense 15μL of solution #2 onto slide #2.
- c. Dispense 15μL of solution #3 onto slide #3.
- d. Dispense 15μL of solution #4 onto slide #4.

14. Wait for 10 minutes and repeat step 13 and wait 10 minutes.
15. Rinse each slide with 5mL of Con-A buffer to remove any unbound Con-A peroxidase. Remove excess buffer by tapping the slide on its side. Allow to air dry on its side.
16. Place the slides on the bench top and dispense 4 drops of Peroxidase Substrate Solution onto each set of cells.
17. Wait 5 minutes and repeat step 16.
18. Gently rinse with DI water. Remove excess moisture by tapping the slide on its side. Allow to air dry.
19. Place a cover slip over slide #1, #2, #3, and #4. Look at the cells under the microscope.
20. Record your results: No color, Light purple, Dark purple.
21. Clean-Up:
- Dispose of your slides in the Biohazards Sharps container.
- Dispose of your toothpicks and disposable microliter pipet tips in the Biohazards Sharps container.
- Dispose of any chemicals in the Chemical Waste container.
- Dispose of any tissue/paper towel with absorbed chemicals in the Biohazards Softs container.
- Dispose of transfer pipets and microcentrifuge tubes in the Biohazards Softs container.

C. Immunohistochemistry-Red Blood Cells
Equipment

• Sheep blood
• Wax pen
• Microscope
• 2 Slides
• 4 Cover slips
• Ethanol
• Microliter pipets
• Transfer pipets
• Small beakers
• Microcentrifuge tubes
• Con-A Buffer (pH 6.8)
• Con-A solution (Con-A lectin and Con-A buffer)
• 1M Galactose (Galactose and Con-A buffer)
• 1M Mannose (mannose and Con-A buffer)
• Erythrocyte suspension (2mL of Con-A buffer and 2 drops of sheep blood)

Protocol

1. Your instructor will prepare the Erythrocyte Suspension (2mL Con-A buffer and 2 drops of sheep blood). An aliquoted of 0.2mL of the suspension will be given to each group. ALWAYS MIX THIS SUSPENSION BEFORE USING.
2. Label 4 microcentrifuge tubes: #1, #2, #3, and #4.
3. Fill each microcentrifuge tube accordingly and mix well.
4. Incubate at room temperature for 30 minutes and shake the microcentrifuge tubes every 5 minutes during this incubation period.
5. You will place two samples on each slide.
6. Label each side of the slide either: #1, #2, #3, or #4.
7. Using a wax pen, draw a circle next to each label. This is where you will place your solutions.
8. Dispense 10μL of the proper solution to each slide. Don’t forget to mix the solution before you transfer it to the slide.
9. Place a cover slip over each solution and examine 100 RBCs and using a microscope. Record the following: hemagglutination, no hemagglutination and % of RBC contact.
10. Clean-Up:
- Dispose of your slides in the Biohazards Sharps container.
- Dispose of your disposable microliter pipet tips in the Biohazards Sharps container.
- Dispose of any chemicals in the Chemical Waste container.
- Dispose of any tissue/paper towel with absorbed chemicals in the Biohazards Softs container.
- Dispose of transfer pipets and microcentrifuge tubes in the Biohazards Softs container.

Immunohistochemistry Post-Lab Assignment: A. Immunohistochemistry-Demo using models
● Draw three examples for the cheek cell demo. Label the cell, antibody, and antigen. Determine if there is specificity and/or competitive inhibition. Determine if the cells will bind the antigen/lectin and result in the cells turning purple.

Draw three examples for the red blood cell demo. Label the cell, antibody, and antigen. Determine if there is specificity and/or competitive inhibition. Determine if the cells will bind the antigen/lectin and result in hemagglutination.

Immunohistochemistry-Cheek Cells
● Draw your results for Slide #1.
o What color were the cells? ____________________________ o
Did competitive inhibition occur? ________________________
Draw your results for Slide #2.
o What color were the cells? ____________________________ o

Did competitive inhibition occur? ________________________
● Draw your results for Slide #3.
o What color were the cells? ____________________________ o
Did competitive inhibition occur? ________________________
● Draw your results for Slide #4.
What color were the cells? ____________________________ o
Did competitive inhibition occur? ________________________

● Explain the mechanism behind the purple color reaction.
● Which sugar inhibited Con A binding to cheek epithelial cells? _____________

Immunohistochemistry-Red Blood Cells ● Draw your results for Slide #1.
o Did hemagglutination occur? __________________________ o Did competitive inhibition occur? ________________________
● Draw your results for Slide #2.
o Did hemagglutination occur? __________________________ o Did competitive inhibition occur? ________________________
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● Draw your results for Slide #3.
o Did hemagglutination occur? __________________________ o Did competitive inhibition occur? ________________________
● Draw your results for Slide #4.
o Did hemagglutination occur? __________________________ o Did competitive inhibition occur? ________________________
● Which sugar inhibited Con A from agglutinating red blood cells? __________

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