INNOVATIVE CELL BIOLOGY AND
IMMUNOLOGY
PRACTICALS
PRACTICAL 1 – SENSITIZATION IN VITRO: EXCISED SKIN TISSUE
SECTIONS, CD1A AND LANGERIN IMMUNOHISTOCHEMICAL
STAINING ON CRYOSECTIONS
BACKGROUND
The skin consists of two co-dependent layers; the outermost epidermis and the
underlying thicker dermis. The epidermis contains keratinocytes, melanocytes,
Merkel cells, Langerhans’ cells and other immune cells. The dermis consists of
collagen, elastin and other matrix proteins. The dermis contains hair follicles,
sweat-, sebaceous and apocrine glands, lymphatic vessels, nerves and blood
vessels. The fibroblast is a major cell type of the dermis.
Human skin is the external barrier to harmful environmental factors. Upon
environmental assault, Langerhans cells (LC) residing in the epidermis migrate
through the dermis towards the
lymphatic vessels.
Migration of LC can be visualized by
immunohistochemical staining (IHC) of
skin sections with CD1a and langerin
antibodies.
The primary antibody specifically
recognizes the protein for which the
staining is performed (Fig 1, Step 1).
The sections will then be incubated
with a secondary antibody that
recognizes the primary immunoglobulin and is coupled to the enzyme horse
radish peroxidase (HRP). We will use Envision, which has a large number of
secondary antibodies and HRP coupled to a dextran backbone (Fig 1, Step 2).
The enzyme HRP converts a substrate (3-Amino-9-ethylcarbazole, AEC) into a
red-coloured substance (Fig 1, Step 3), visible under the microscope. The larger
amount of HRP coupled onto the protein of interest will enhance the staining
compared to “traditional” HRP staining methods like avidin-biotin complex
method (ABC).
Controls are very important for IHC. Proper controls are needed to confirm the
validity of the staining pattern and to exclude experimental artefacts. Basic
controls are:
- Positive control: a section from a tissue known to express the protein of
interest.
- Negative control: a section from a tissue known not to express the target
antigen.
- Endogenous tissue background control: the immunohistochemical
procedure in which the primary antibody is not applied or an isotype
antibody (which does not recognize the antigen of interest) is applied to
the tissue which does express the protein of interest.
In this practical course we will include the positive control and IgG1 control.
, In this practical course, we aim to use immunohistochemical stainings to
determine whether the skin has been exposed to a harmful chemical which has
penetrated the stratum corneum of the epidermis (Key event 1 in AOP
sensitization).
PRACTICAL
Preparations of reagents and solutions
Dilutions
Slid Sample Primary antibody Secondary
e antibody
Section 1 Section 2
1 Skin CD1a Langerin Envision
2 Skin IgG1 IgG1 Envision
3 Skin exposed to vehicle CD1a Langerin Envision
(water)
4 Skin exposed to CD1a Langerin Envision
sensitizer
5 Skin exposed to irritant CD1a Langerin Envision
CD1a: 75 μl per slide → 4 slides = 4x75 = 300 μl + 50 μl extra → 350 μl dilution
1:30 CD1a
Langerin: 75 μl per slide → 4 slides = 4x75 = 300 μl + 50 μl extra → 350 μl
dilution 1:25 Langerin
IgG1: 75 μl per slide → 2 slides, but different ratio’s – 2x 125 μl dilution 1:30
IgG1 1x, 1:25 IgG1 1x
Needed
- CD1a: 350/30 = 11.67 μl needed
- Langerin: 350/25 = 14 μl needed
- IgG1 first: 125/30 = 4.17 μl needed
- IgG1 second: 125/25 = 5 μl needed
QUESTIONS
1. Which cells and what part of these cells do the antibodies used in this
practical course stain (CD1a, Langerin and IgG1).
a. CD1a: CD1a stains dendritic cells in general, not only
Langerhans cells. It is a marker for all, it activates a receptor
on the dendritic cells.
b. Langerin: Langerin stains specific dendritic cells, the
Langerhans cells only
Both CD1a and Langerin stain external, so the outside of the
cell – staining of surface proteins. But due to the sections that
are cut (5 micrometers) are smaller than the cell itself, the cell
is cut in half and therefore the inside is also stained, because
granules on the inside also present the receptors.
c. IgG1: supposed to stain nothing, if the staining is negative it
can be assumed the signal of the other antibodies (Langerin
and CD1a) is valid, because the signal is not produced by other
proteins.
2. Which cells and what part of these cells do you expect to stain with
haematoxylin?
IMMUNOLOGY
PRACTICALS
PRACTICAL 1 – SENSITIZATION IN VITRO: EXCISED SKIN TISSUE
SECTIONS, CD1A AND LANGERIN IMMUNOHISTOCHEMICAL
STAINING ON CRYOSECTIONS
BACKGROUND
The skin consists of two co-dependent layers; the outermost epidermis and the
underlying thicker dermis. The epidermis contains keratinocytes, melanocytes,
Merkel cells, Langerhans’ cells and other immune cells. The dermis consists of
collagen, elastin and other matrix proteins. The dermis contains hair follicles,
sweat-, sebaceous and apocrine glands, lymphatic vessels, nerves and blood
vessels. The fibroblast is a major cell type of the dermis.
Human skin is the external barrier to harmful environmental factors. Upon
environmental assault, Langerhans cells (LC) residing in the epidermis migrate
through the dermis towards the
lymphatic vessels.
Migration of LC can be visualized by
immunohistochemical staining (IHC) of
skin sections with CD1a and langerin
antibodies.
The primary antibody specifically
recognizes the protein for which the
staining is performed (Fig 1, Step 1).
The sections will then be incubated
with a secondary antibody that
recognizes the primary immunoglobulin and is coupled to the enzyme horse
radish peroxidase (HRP). We will use Envision, which has a large number of
secondary antibodies and HRP coupled to a dextran backbone (Fig 1, Step 2).
The enzyme HRP converts a substrate (3-Amino-9-ethylcarbazole, AEC) into a
red-coloured substance (Fig 1, Step 3), visible under the microscope. The larger
amount of HRP coupled onto the protein of interest will enhance the staining
compared to “traditional” HRP staining methods like avidin-biotin complex
method (ABC).
Controls are very important for IHC. Proper controls are needed to confirm the
validity of the staining pattern and to exclude experimental artefacts. Basic
controls are:
- Positive control: a section from a tissue known to express the protein of
interest.
- Negative control: a section from a tissue known not to express the target
antigen.
- Endogenous tissue background control: the immunohistochemical
procedure in which the primary antibody is not applied or an isotype
antibody (which does not recognize the antigen of interest) is applied to
the tissue which does express the protein of interest.
In this practical course we will include the positive control and IgG1 control.
, In this practical course, we aim to use immunohistochemical stainings to
determine whether the skin has been exposed to a harmful chemical which has
penetrated the stratum corneum of the epidermis (Key event 1 in AOP
sensitization).
PRACTICAL
Preparations of reagents and solutions
Dilutions
Slid Sample Primary antibody Secondary
e antibody
Section 1 Section 2
1 Skin CD1a Langerin Envision
2 Skin IgG1 IgG1 Envision
3 Skin exposed to vehicle CD1a Langerin Envision
(water)
4 Skin exposed to CD1a Langerin Envision
sensitizer
5 Skin exposed to irritant CD1a Langerin Envision
CD1a: 75 μl per slide → 4 slides = 4x75 = 300 μl + 50 μl extra → 350 μl dilution
1:30 CD1a
Langerin: 75 μl per slide → 4 slides = 4x75 = 300 μl + 50 μl extra → 350 μl
dilution 1:25 Langerin
IgG1: 75 μl per slide → 2 slides, but different ratio’s – 2x 125 μl dilution 1:30
IgG1 1x, 1:25 IgG1 1x
Needed
- CD1a: 350/30 = 11.67 μl needed
- Langerin: 350/25 = 14 μl needed
- IgG1 first: 125/30 = 4.17 μl needed
- IgG1 second: 125/25 = 5 μl needed
QUESTIONS
1. Which cells and what part of these cells do the antibodies used in this
practical course stain (CD1a, Langerin and IgG1).
a. CD1a: CD1a stains dendritic cells in general, not only
Langerhans cells. It is a marker for all, it activates a receptor
on the dendritic cells.
b. Langerin: Langerin stains specific dendritic cells, the
Langerhans cells only
Both CD1a and Langerin stain external, so the outside of the
cell – staining of surface proteins. But due to the sections that
are cut (5 micrometers) are smaller than the cell itself, the cell
is cut in half and therefore the inside is also stained, because
granules on the inside also present the receptors.
c. IgG1: supposed to stain nothing, if the staining is negative it
can be assumed the signal of the other antibodies (Langerin
and CD1a) is valid, because the signal is not produced by other
proteins.
2. Which cells and what part of these cells do you expect to stain with
haematoxylin?
