Staining Normal Skin

Allen MillerUCLA, Modlin Laboratory

Department of ImmunologyDivision of Dermatology

8-15-06

Modlin LaboratoryThis past summer I spent two months working in an

Immunology laboratory with a group of undergraduates, medical students, PhD’s and MD’s. The laboratory was run by a dermatologist named Robert Modlin. Dr. Modlin is a leading researcher in the field of Immunology with numerous publicationsin journals such as Nature Medicine. During my time at the laboratory, Dr. Modlin was working on several projects involvingresearch of the lepromatous and tuberculoid forms of leprosy. Hewas especially interested in researching the affects of the Mycobacterium Leprae (M. Leprae) on macrophages and dendritic cells. I was mentored and trained by Dr. Modlin’s colleague, Dr. Maria Teresa Ochoa. Dr. Ochoa is a clinical dermatologist as well as a researcher. Under Dr. Ochoa, I performed numerous experiments on normal skin using Immunohistochemistry and Immunofluorescent staining. Staining normal skin allowed me to compare healthy skin tissue to the lesions found in leprosy.

Immunohistochemistry

• The first staining experiment I did involved the identification of several different cells via immunohistochemistry.

• Specifically, I used the Avidin Biotin Complex (ABC System) protocol.

• I did the same experiment on the breast tissue of two different individuals who had normal skin.

Background

• Immunohistochemistry involves the localization of antigens on the surface of cells in tissue sections by use of labeled, antigen-specific antibodies.

• The antigen-antibody interactions are visualized by markers such as enzymes or colloidal gold.

• Immunohistochemistry allows us to search for one specific antigen in each section of skin tissue.

Immunohistochemistry: Step 1• The primary antibody

binds to its specific antigen. In reality the antigens are much smaller and greater in quantity.

• All of the antibodies I used were Cluster of Differentiation (CD) Antigens.

• The primary antibodies attached to cells in both the Dermis and the Epidermis.

Epidermis

Dermis

Antigens (either withinthe cell or on the surface)

Primary Antibody

Step 2• The antibody-antigen

complex is bound by a secondary antibody.

• I used a biotinylated anti-mouse as my secondary antibody.

Epidermis

Dermis

Antigens (either withinthe cell or on the surface)

Primary Antibody

Secondary Antibody

Step 3

• A complex of avidin biotin peroxidase (ABC) is used to amplify and solidify the staining.

Epidermis

Dermis

Antigens (either withinthe cell or on the surface)

Primary Antibody

Secondary Antibody

ABC

Step 4

• The 3-amino-9-ethyl-carbazole (AEC) substrate is added forming a colored deposit at the antibody-antigen site.

• The AEC substrate leaves a reddish-brown color that will be seen in my slides.

Epidermis

Dermis

Antigens (either withinthe cell or on the surface)

Primary Antibody

Secondary Antibody

ABC

AEC

Photographic Results

• All of the immunohistochemistry slides were photographed with a conventional compound optical microscope camera that ranged in power from 10x to 40x.

• The compound optical microscope uses a system of lenses to magnify images of small samples.

• Light is controlled and focused by a condenser with a plano-convex lens located at the base.

• The different individual’s skin sections were numbered 14153B and 11151B.

Langerin

• Langerhan cells, which are the precursors to dendritic cells, express langerin.

• Starting out as immature dendritic cells, Langerhan cells constantly sample their surrounding areas for pathogens like bacteria using their toll-like receptors (TLR’s).

• When pathogens invade skin tissue, the langerhan cells process the pathogens and then travel to the T-cell section of the lymph node where they mature into dendritic cells.

14153B

11151B

CD14 (zymed)• CD14 is expressed in monocytes,

dendritic cells, activated B cells and especially in macrophages.

• The stained cells to the left are dendritic cells located in the dermis.

• Dendritic cells are important immune cells present in skin. Once activated, these antigen presenting cells present their antigen fragments, which are derived from pathogens, to T and B lymphocytes.

• The zymed brand of CD14 is an IgG2b isotype.

14153B

11151B

CD14 (pharmingen)

• The pharmingen brand of CD14 is an IgG2a isotype.• The dendritic cells above are located mainly in the dermis;although,

there are a few cells in the epidermis.

11151B 11151B

14153B

CD1a (Dako)

CD1a is a marker for antigen presenting cells (APC’s), especially dendritic cells migrating from the epidermis. APC’s, which also include macrophages, present foreign antigens to T cells that exhibit specificity towards a certain antigen. APC’s are the only cells able to activate a helper T cell that has never encountered its antigen.

CD1b

11151B

•Leprosy is an infectious disease that damages the skin, nerves and mucous membrane.•The two forms of leprosy are the tuberculoid form and the lepromatous form.•The tuberculoid form is less severe and can be eradicated with treatment.•The lepromatous form progresses and never lessens in intensity.•Patients with the lepromatousform of leprosy, associated withabsent M. leprae specific cell mediated immunity, do not express CD1 antigens.

•CD1b is another APC marker in normal skin.

DC sign (CD 209)

• Formally it was thought that dendritic cells express DC-sign. It is now believed that a subset of macrophages express DC-sign.

• In patients with the lepromatous form of leprosy, the Modlin Laboratory discovered CD1b+ dendritic cells were not found but, DC-sign+ macrophages were found in lesions.

• Since macrophages were positive for a dendritic cell marker the question brought up is: Are dermal dendritic cells macrophages?

• The Modlin laboratory believes the answer is: yes.

14153B 11151B

CD68• Macrophages, such

as the ones to the right, as well as some T cells present in lymph nodes express CD68.

• Macrophages are APC’s but, they also act as phagocytes.

• The CD68 antigen is mainly located in lysosomes within the macrophage.

• Lysosomes contain degrading enzymes for breaking down ingested pathogens during phagocytosis.

14153B

14153B

Next step: Immunofluorescence

Immunofluorescence

•The next staining experiment I did was very similar to Immunohistochemistry.•Immunofluorescence is a more advanced method for the detection of cells. Confocal imaging allows the results of immunofluorescent staining to be be visualized better. •I double stained normal breast tissue using markers for both dendritic cells and macrophages.

Background

•Immunofluorescent staining allows the localization of different antigens within a single section of skin tissue by using multiple primary antibodies.•The antigen-antibody interactions are visualized by fluorescent dyes of different colors.•Staining for two different cells will work as long as each antibody is of a different isotype.•Thus immunofluorescence builds on immunohistochemistry in that we can stain for more cells as well as visualive our stainings better.

Immunofluorescence: Step 1

• The primary antibody binds to its specific antigen.

• The primary antibodies I used were all dendritic cell markers

• The primary antibodies attached to cells mainly in the epidermis.

Epidermis

Dermis

Antigens

Primary Antibody

Step 2• The antibody-antigen

complex is bound by a secondary antibody.

• I used a fluorescent dye named Alexafluor 488 as my secondary antibody.

• The number 488 is a reference to 488 nanometers which is the light wavelength needed when trying to view the antibody under a confocal microscope.

• On the electromagnetic spectrum segment of visible light, 488 nm is a green light.

Epidermis

Dermis

Antigens

Primary Antibody

SecondaryAntibody

Step 3• A second primary antibody is

added, binding to its specific antigen

• The second primary antibodies I used were all macrophage markers.

• The macrophages I stained for were in the dermis.

Epidermis

Dermis

Antigens

Primary Antibody

SecondaryAntibody

2nd Primary Antibody

Step 4• A second secondary

Antibody binds to the antigen-antibody complex.

• I used Alexafluor 568 (568 nanometers) which is the color red on the electromagnetic spectrum.

Epidermis

Dermis

Antigens

Primary Antibody

SecondaryAntibody

2nd PrimaryAntibody

2nd Secondary Antibody

Step 5• The last step involves

staining the nucleus of all the cells (not just the tagged cells) with DAPI.

• DAPI is short for 6-diamidino-2-phenylindole

• DAPI binds strongly to the DNA in the nucleus of cells without overlaping other fluorescent dyes.

• DAPI has a 358 nm wavelegth and thus appears blue.

Epidermis

Dermis

Antigens

Primary Antibody

SecondaryAntibody

2nd PrimaryAntibody

2nd Secondary Antibody

Confocal Laser Scanning Microscopy• Confocal imaging is a technique used to

increase micrograph contrast and create three dimensional reconstructions.

• The key function a confocal microscope can perform is the production of blur-free images of thick specimen at various depths.

• Whereas the conventional microscope floods the entire surface of a specimen (allowing for only a “bird’s eye” two dimensional view), a confocal microscope uses a spatial pinhole to narrowly focus the laser rays and fluorescent lights producing a high resolution image.

• Confocal laser microscope scanners take two dimensional photographs at different layers on the sample before superimposing all of them on top of each other for the final image.

Top of skin sample

Top of skin sample

]layers

ConfocalMicroscopeimage

Conventional Microscope image

Confocal Photographic Results

• When scanning my slides with the confocal scanner, I used objective lenses that ranged from 20x to 100x.

• All of the green dyed cells are dendritic cells.• All of the red dyed cells are dermal dendritic cells

thought to be macrophages.• The nuclei of all cells are dyed blue.

Important Antibodies

• Dendritic Cell markers:– *DC lamp– *CD1a, b, c– Langerin– Fascin– CD83– FXIIIa

*antibodies I used in my experiment

?still under research

• Macrophage markers:– *Dcsign/CD 209– L-sign/CD299?– CD64– CD16– CD14– CD68– CD206/MMR?

Normal Skin

Merge

CD1a

CD209

DAPI

Normal Skin

CD1a CD209 CD1a+ CD209 +DAPI

Normal Skin

Merge

CD1a

CD209

DAPI

Normal Skin

CD1a CD209 CD1a+ CD209 +DAPI

Normal Skin

Merge

CD1b

CD209

DAPI

Normal Skin

Merge

DClamp

CD209

DAPI

A very focused Macrophage.

DC sign posotive macrophages (green) perform phagocytosis on M. Leprae (red).

Leprosy Lesion

Notes and Future GoalsThis slide show presentation was researched and put together by Allen Miller. All diagrams made, experiments performed and photographs taken were done by Allen Miller under the mentorship and supervision of Dr. Maria Teresa Ochoa. In the future, I would like to continue research in the field of immunology. Pieces are stilling missing for such puzzles as Leprosy, Human Immunodeficiency Virus (HIV) and Cancer. With the knowledge we have today, with the technology available to our generation, with the amount of researchers spread out across the globe, I believe we can make unparalleled advancements towards the eventual cure of such parasitical diseases.