Immunohistochemical Study of Antibodies
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1 Immunohistochemical Study of Antibodies Alexander Pfeifer 2014
Transcript of Immunohistochemical Study of Antibodies
1
Immunohistochemical Study of
Antibodies
Alexander Pfeifer
2014
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Special thanks to:
Doris Eichelberg, Dominique Eichelberg, Philippe Pfeifer and the BMA for supporting
this project with the needed knowledge, materials and methods.
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Index
.................................................................................................................................... 1
Abstract ........................................................................................................................ 6
Abbreviations ................................................................................................................ 6
Introduction .................................................................................................................. 7
Immunohistochemistry [36] ........................................................................................... 7
Digestive system of a pig [1,3,8] ..................................................................................... 7
Structure of the gastrointestinal wall [1,3,6,8,10,19] ............................................................. 8
Antibodies [7,12,13,18] ...................................................................................................... 9
Monoclonal antibodies [5,17,20] ..................................................................................... 10
Materials and Methods ................................................................................................. 11
A) Chemicals ............................................................................................................ 11
Working Equipment [34] ........................................................................................... 12
B) Buffers and solutions [34] ........................................................................................ 12
10x PBS:............................................................................................................... 12
0.15 M Sodium azide: ............................................................................................ 12
Acetate buffer: ...................................................................................................... 12
AEC stock solution: ................................................................................................ 12
AEC substrate: ...................................................................................................... 13
C) Procedures .......................................................................................................... 13
Preparation of tissue samples ................................................................................. 13
Staining method 2 .................................................................................................. 14
Indirect Staining Method 2, 4 ................................................................................... 14
D) Staining the Tissue [34] .......................................................................................... 15
Results [10,11] ................................................................................................................ 17
Antibodies ................................................................................................................ 18
C IV 22[24]: anti Collagen IV .................................................................................... 18
C-3D5[25]: anti Trefoil Factor ................................................................................... 19
HM 19/2: anti Smooth Muscle ................................................................................. 20
KG 7/30 [26]: anti Endothelial Cells ........................................................................... 21
Lu-5 [27]: anti Cytokeratin........................................................................................ 22
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PB1 [28]: anti Epithelial Basement ............................................................................ 23
PE1 [29]: anti Endothelial Cells ................................................................................. 24
PG1: anti Goblet Cells ............................................................................................ 25
PM1 [30]: anti Neutrophils ....................................................................................... 26
PM-2K [31]:anti Macrophage..................................................................................... 27
PP1 [32]: anti Thrombocytes and Endothelial Cells ...................................................... 28
PP2 [33]: anti Thrombocytes and Endothelial Cells ...................................................... 29
PBS Test ............................................................................................................... 30
Discussion ................................................................................................................... 31
C-IV-22 [15,25] ......................................................................................................... 31
C-3D5 [25] .............................................................................................................. 31
HM 19/2 ............................................................................................................... 31
KG 7-30 [26] ........................................................................................................... 32
Lu-5 [16,27] ............................................................................................................. 32
PB1 [28] ................................................................................................................. 32
PE1 [29] ................................................................................................................. 32
PG1 [] ................................................................................................................... 32
PM1 [22,30].............................................................................................................. 32
PM-2K [31] ............................................................................................................. 33
PP1 [32] ................................................................................................................. 33
PP2 [33] ................................................................................................................. 33
References .................................................................................................................. 34
Appendix ..................................................................Fehler! Textmarke nicht definiert.
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Abstract
The purpose of this paper was to investigate the reactivity of various antibodies with porcine
intestinal tissue. Twelve different antibodies on three different sections of the small intestine
were compared. Some antibodies were originally directed against human tissue, namely
CIV22, C-3D5, HM19/2, KG 7/30, Lu-5 and PM-2K. The other clones PB1, PE1, PG1, PM1,
PP1 and PP2 have been developed against porcine tissue. The antibodies developed against
human tissue still work for swine tissue because the epitope (the motif recognized by the
antibody) is the same in both species. Testing was done on the twelve following
preparations:
Duodenum, lengthwise and crosswise
Jejunum, lengthwise and crosswise
Ileum, lengthwise and crosswise
Every tissue probe was sectioned with a cryostat, brought onto a slide and fixed there. This
was followed by indirect (two-step) staining with twelve different antibodies, each one in
three different concentrations. Hereby the method of indirect staining was used. First a
specific antibody is applied, after which a secondary antibody carrying the color-giving
peroxidase was added. The secondary antibody docks onto the primary antibody, marking
the cell or tissue structure. Through observation under a light microscope, the specific
staining attributes of the antibodies and the quality of staining could be compared with each
other. The results and discussion in this paper will focus on the jejunum cross sections, since
it yielded the best results.
This work was performed entirely by Alexander Pfeifer, with material support from BMA
Biomedicals, a division of Chemoforma AG. This publication is accessible through
www.bma.ch and may be freely distributed citing ©Alexander Pfeifer.
Abbreviations
HRPO: Horseradish peroxidase
PBS: Phosphate Buffered Saline
AEC: Aminoethyl carbazol
NRS: Normal Rat Serum
NMS: Normal Mouse Serum
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Introduction
Immunohistochemistry [36]
In immunohistochemistry, specified antigens are stained by antibodies labeled with a color
giving substance. The stained cells or structures can then be observed through a microscope
and allow for the study of the microscopic anatomy of a tissue, its organization and function.
The word “immuno” stands for the antibodies involved in the process and “histo” for the
tissue being treated.
Digestive system of a pig [1,3,8]
The digestive system of a pig is similar to that of a human. Food is transported through the
esophagus into the stomach. Here, the food gets mixed with hydrochloric acid and enzymes
secreted by gastric glands. This lowers the pH value to 1.5-2.5, killing bacteria in the food.
Before leaving the stomach, the ingested food gets covered in mucus to prevent damage to
the small intestine by the low pH value. Upon leaving the stomach the chyme now enters the
duodenum, the first section of the small intestine, which is followed by the jejunum and the
ileum. The small intestine is the major site of nutrient absorption. It is lined with sodium
bicarbonate producing cells, to provide alkalinity and protect the intestine through increasing
the pH value. The pancreas discharges digestive enzymes into the small intestine to help
break up protein, fat and carbohydrates. Additionally, the liver produces bile which is also
added to the chyme. The bile salts emulsify and allow for the absorption of fat. Once the
chyme is mixed with these juices, the actual absorption of nutrients begins and continues
throughout the jejunum and ileum. The small intestine is lined with villi. These are finger-like
projections that protrude out of the epithelial layer. They are between 0.5-1.5 mm long and
are covered with microvilli. They form the so-called brush border through which nutrients are
absorbed. Through the microvilli, amino acids and simple sugars are absorbed and are led
through the villi into the circulatory system. Fats are also absorbed but enter the lymphatic
system. The chyme is still in a very liquid form when it leaves the small intestine, especially
because more liquid has been added and hardly any absorbed. The large intestine now
absorbs excess water from the chyme. Not many nutrients are absorbed anymore since most
of them were already absorbed by the small intestine. Altogether, the upper intestine
secretes around six liters of liquid per day, which are absorbed in the lower part.
Since the lumen of the gastrointestinal tract comes in contact with material from the external
environment, it is important to have an extra immune response within the digestive tract.
The Peyer’s patches[23] are an important part of this function. They generate an additional
immune response within the mucosa and deliver supplementary protective leukocytes
(macrophages, dendritic cells, B-lymphocytes, and T-lymphocytes.) Peyer’s patches are
mostly located in the ileum, which is how it can be differentiated from the duodenum and
the jejunum.
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Structure of the gastrointestinal wall [1,3,6,8,10,19]
The gastrointestinal wall consists of four concentric layers which are relatively consistent
throughout the intestinal tract. The inner layer facing the lumen is the mucosa, directly
underneath lies the submucosa. These two layers are surrounded by a muscle layer, the
muscularis externa, which helps propel the food forwards. The serosa is the outermost layer
representing the border of the gastrointestinal tract.
The mucosa can be further differentiated into three layers: the epithelium, the lamina
propria and the muscularis mucosae. Throughout the gastrointestinal tract, the epithelium
varies the most in its form of appearance. Most of the absorbing processes take place in the
epithelium such as in the small intestine. Here, the epithelium creates the brush border of
villi.
Figure 1 Cross-section through the gastrointestinal wall. Larger Tissue structures have been colored in and numbered.
1) Longitudinal muscle (muscularis externa) 5) Muscularis mucosae
2) Sphincter muscle (muscularis externa) 6) Epithelium with villi and intestinal crypts
3) Submucosa 7)Lumen
4) Blood vessel (submucosa)
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Antibodies [7,12,13,18]
Antibodies belong to the specific acquired immunity. Other than our innate immune system,
which we are born with, we acquire specific immunity throughout our life. Harmful organisms
that get past our innate immune system are stopped in different ways. This is where the
specific immunity takes over. An antibody or immunoglobulin (Ig) is a globular protein. It’s
produced by circulating leukocytes called plasma cells. It marks harmful substances and
organisms to be recognized and taken care of by our immune system. The antibody carries
two identical regions which can recognize and bind to a fitting antigen. This side of the
antibody is the recognizing side. The antigen to which the antibody binds is mostly a surface
structure on a protein such as a group of a few sugars or amino acids. When antibodies bind
to an antigen, a mechanism is triggered and more antibodies of that type are produced.
Antibodies in our body are directed to recognize mostly foreign and toxic substances and not
to recognize antigens belonging to our own body. If antibodies mark the body’s own tissue,
it will result in an autoimmune disease. Every antibody has a specific antigen to which it
binds, very similar to a key and lock. That’s why such a large variation of antibodies exists:
to ensure that all the different surface structures can be detected. The part of the antibody
that connects to the antigen is called an antigen binding fragment. The exact spot on the
antigen to which the antibody binds is called the epitope. Antibodies also appear in different
forms in our body, called isotypes. IgM antibodies are produced as pentamers. They are part
of the primary response to infection or immunization and can later switch to one of the other
isotypes. The most widespread is IgG. It shows high affinity when binding to antigens. It is
also the major immunoglobulin synthesized during a secondary immune response. Its
primary function is to neutralize bacterial toxins and mark microorganisms to enhance their
phagocytosis, i.e. their destruction through ingestion by specialized white blood cells. IgA
appears in seromucous secretions such as saliva, tears, nasal fluids, sweat and the
secretions of the gastrointestinal tracts, to which the small intestine belongs. It defends
these surfaces that come in contact with external material against the attack of
microorganisms or toxins. IgA is mainly secreted as a dimer. Our body produces daily forty
milligrams per kilogram body weight. This is more than any other immunoglobulin, since a
lot of it is excreted directly out of our body. IgD controls the activation and suppression of
lymphocytes, regulating the production of antibodies this way. IgE occurs in small quantities
in our body and is responsible for allergic reactions. By binding to mast cells and
concomitant release of histamine, typical allergy symptoms such as itching and tearing are
induced.
Figure 3
Figure 2 [35] The different isotypes of an antibody
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Monoclonal antibodies [5,17,20]
A monoclonal antibody is the product of one single antibody producing cell. Initially, the
antigen against which it is directed is determined. This antigen is then isolated from a
natural source, or synthesized chemically. For example, when an antibody against muscle
requires isolating an antigen specifically found in muscle tissue. This antigen is then injected
into a host animal. The mouse is a frequently used host species because the immunogen
often originates from human or species other than mouse. Upon immunization, the host’s
specific lymphocytes start to proliferate and produce antibodies against the immunized
antigen. In parallel, a culture of myeloma[21] cells is brought up, which is a cell line that
divides and proliferates eternally. Then the spleen of the mouse is removed and spleen cells
are extracted. The spleen cells of the mouse are now fused with the myeloma cells. A
minority of 1:1 fusion products results in permanent proliferation. The process up to this
point may take 4 to 6 months. Now the fused cells are brought into a culture medium in
which only myeloma / spleen cell fusions, so called hybridomas, survive. These hybridomas
are then diluted and put in microtiter wells so that, statistically, one cell is in one well. Now
the microtiter wells are screened for the presence of the desired antibody and wells
containing none are discarded. Once all the wells containing an antibody are found, they are
cultured and screened once again. The clones that have been produced by the parent cells
are now again diluted and spread across microtiter wells. This process called recloning
ensures a minimal variety of fused hybridoma cells; they all descend from one single mother
cell and can be considered identical twins. After letting the cells proliferate once again they
are then tested for quantity and quality of the antibody and the best of them is chosen. This
one is once again put in a suitable culture medium in which it reproduces. These steps may
take another two to three months. Once a large enough quantity has been produced, the
fluid containing the antibodies needs to be purified. Cells, cell debris, unwanted proteins and
other impurities have to be removed. By centrifugation and filtration most of the particulate
impurities are removed. Affinity chromatography9 is one of the methods to separate the
antibody from the remaining impurities. Hereby the fluid containing the antibody is poured
through a gel matrix. The gel matrix will have an affinity towards the antibody so that
everything except the antibody will be flushed through the gel matrix. Now the gel matrix
can be washed and then the antibodies are eluted with an appropriate solvent.
Figure 3 screening of a microtiter well plate. Wells where an antibody is present are stained yellow.
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Materials and Methods
A) Chemicals
Acetic acid CH3COOH: Fluka 45731
AEC 3-Amino-9-Ethylcarbazol C14H14N2: Sigma A-5754
Dimethylformamide C3H7NO: Fluka 40250
di-Sodiumhydrogenphosphate dihydrat Na2HPO4: Fluka 71638
Hydrogen peroxide H2O2: Fluka 95300
Mayer’s Hemalum: Fluka 51275
Monoclonal C IV 22: BMA, T-1201
Monoclonal C-3D5 : BMA, T-1451
Monoclonal HM19/2 : BMA, T-1203
Monoclonal KG 7/30: BMA, T-1104
Monoclonal Lu-5 : BMA, T-1302
Monoclonal PB1: BMA, T-3504
Monoclonal PE1: BMA, T-3510
Monoclonal PG1 : BMA, T-3509
Monoclonal PM1: BMA, T-3503
Monoclonal PM-2K : BMA, T-1051
Monoclonal PP1: BMA, T-3506
Monoclonal PP2: BMA, T-3507
Sodium azide NaN3: Fluka 71290
Normal Goat Serum NGS: Gibco BRL
Normal mouse serum NMS: JacksonImmunoresearch
Normal rat serum NRS: BMA Biomedicals
Peroxidase conjugated Goat-anti-Mouse IgG (H+L) HRPO
(Jackson ImmunoResearch, 115-035-146)
Peroxidase conjugated Goat-anti-Mouse IgM µ Chain Specific
(Jackson ImmunoR esearch, 115-035-020)
Peroxidase conjugated Goat-anti-Rat IgG+IgM (H+L)
(Jackson ImmunoResearch, 112-035-068)
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Potassium chloride KCl: Fluka 60130
Potassiumdihydrogenphosphate KH2PO4: Fluka 60220
Royalmount: BMA Biomedicals, T-3402
Sodium chloride NaCl: Fluka 71381
Sodium hydroxide NaOH: Sigma 30620-1KG-R
Tissue-Tek® O.C.T™ Compound and Cryomolds®: Sakura Finetek Europe
Working Equipment [34]
The following Equipment is needed for this experiment:
Cryostat, microscope, glass cuvettes, pipettes, paper filters, graduated cylinders, funnels,
vacuum pump, heating plate, ventilator, humid chamber, Q tips, Kleenex, grease pen,
diamond pencil, cover slips, timer
B) Buffers and solutions [34]
Buffers and solutions were made according to the instruction of the BMA Biomedicals
instructions for staining fresh frozen tissue sections.
10x PBS:
KCl (m.w. 74.6): 1.6g/l
KH2PO4 (m.w. 136): 2.8g/l
Na2HPO4x2H2O (mw 178): 13.4g/l
NaCl (mw 58.4): 77.8g/l
Dilute 1/10 with distilled water before use, check pH and adjust to pH 7.2 if necessary
0.15 M Sodium azide:
Dissolve 4.88g NaN3 in 500ml PBS pH 7.2.
Acetate buffer:
Dissolve 1.4 ml acetic acid in 250 ml distilled water, adjust pH to 4.9 with 10M NaOH.
Store at 2-8°C for up to two days.
AEC stock solution:
Dissolve 0.4g 3-Amino-9-Ethylcarbazol in 100ml Dimethylformamid
Store at 2-8°C for up to four weeks
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AEC substrate:
5ml AEC stock solution
95ml acetate buffer, pH 4.9
Filter through paper filter and add 40µl 30% H2O2 immediately before use.
C) Procedures
Preparation of tissue samples
Small intestines of a pig were collected at the slaughterhouse in Basel. Approximately twenty
centimeters each of the duodenum, the jejunum and the ileum were retrieved. They were
packed separately and immediately submerged in cold PBS for transport. After that,
rectangles in the dimensions of one by two centimeters were cut out of the tissue, once
lengthwise and once crosswise. The cuts were then folded in half lengthwise so that the
inside of the swine intestine faced inward. Using Tissue-Tek® O.C.T™ Compound, the tissue
samples were then fixed onto a freezing plate so that they were standing, and snap-frozen in
the cryostat. Once completely solid, the tissue samples were mounted in the cryostat for
cutting. Cuts were made at -22°C and 5µm thickness. 36 sections were produced of every
tissue sample and an extra one for a negative control. Three sections of the same tissue
sample were placed on one slide. The fresh sections were left to dry in the air for at least
twenty minutes before submerging them in ice cold acetone for ten minutes and then letting
them air dry overnight. This way the tissue was now fixed. Fixation ensures that there is
almost no water left in the tissue so that no ice crystals develop when the sections are
stored before further handling. The slides were carefully wrapped in aluminum foil and
stored in a freezer at -22°C.
Figure 4 Orientation of the one by two centimeter tissue sections
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Staining method 2
There are several staining methods available, all of them having their advantages and
disadvantages. Aspects that should be brought into consideration before choosing an
appropriate staining method would be:
Type of Specimen
Sensitivity needed
Time
Cost
For our purposes, an indirect staining method is suitable. It is not too complicated and still
offers satisfactory results.
Indirect Staining Method 2, 4
After an initial treatment to prevent non-specific reactions, the primary antibody is added. It
binds with the antigens on our tissue. Thereafter, the secondary antibody is added. It is
directed towards the primary antibody and binds accordingly. A peroxidase is attached to the
secondary antibody. Upon adding the chromogenic AEC stock solution, it reacts with the
peroxidase, forming a red end product.
The peroxidase is an enzyme isolated
from the root of the horseradish plant. It
stays inactive during the absence of a
dye. But when one is added, the
peroxidase reacts with it causing the dye
to oxidize and change color. There are
several dyes that can be used for this
application. Their chromogenic and
insoluble properties are desirable
attributes that make them useful for
immunohistochemistry. For my
experiment I used AEC (3-Amino-9-
ethylcarbazole). Upon oxidation, AEC
becomes insoluble and turns red. The red
coloring marks the targeted antigens and
makes it possible for us to observe
distribution through a light microscope.
Figure 5 indirect method: primary antibody attached to tissue antigen. Secondary antibody on primary with peroxidase.
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D) Staining the Tissue [34]
This Step was also done according to the BMA Biomedical instructions for staining fresh
frozen tissue sections.
1. Remove the slides from the freezer and take away the aluminum foil after five minutes.
Dry the sections for 30 minutes in an air stream. (Ventilator only, no heat)
2. Dip slide 1x5 minutes in PBS, pH 7.2.
3. Blocking of endogenous peroxidase:
Mix 100ml 0.15M Sodium azide and 0,5 ml 30% H2O2 in cuvette, place sections in holder,
and incubate for 20 minutes at room temperature. Rinse 1x with PBS and incubate for 1x5
minutes in PBS, pH 7.2 (100ml cuvette)
Peroxidase is a naturally occurring enzyme that is found in many tissues. This step serves to
suppress peroxidase activity that is not specifically associated with the detection antibody.
4. Blocking with serum:
Coat entire tissue sections with 10% NGS in PBS, incubate for 30 minutes at room
temperature in a humid chamber.
This step serves to block non-specific binding of the antibody to the tissue.
5. Remove serum by careful aspiration. Do not wash any further.
6. Incubation with primary antibody:
Apply primary antibody. Incubate for 1-16 hours depending on antibody (ab) in humid
chamber. Incubation time varies from antibody to antibody.
Specific binding of the antibody may be enhanced by prolonged exposure. On the other
hand, non-specific binding may be minimal at short incubation times.
Important: include positive and negative control
7. Remove antibody solution by aspiration and rinse 1x in PBS, then wash for 1x5 minutes in
large cuvette with PBS pH 7.2
8. Incubation with peroxidase - conjugated secondary antibody
Prepare the following solution:
Peroxidase-conjugated secondary antibody made according to your own titration or
manufacturer's suggestion.
Normal Goat Serum: 10% (Normal Serum of tissue species; (5%) this step is required if the
secondary antibody serum is not absorbed) PBS Add to 100% Incubate with 100-500µl per
section in humid chamber for 60 minutes. This step detects and enhances the presence of
primary antibody.
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9. Aspirate antibody solution and rinse 1x in PBS. Wash in PBS for 1x5 minutes in large
cuvette (PBS pH 7.2)
10. Incubation with AEC substrate:
Incubate with filtered AEC substrate solution (see above) for 12 minutes in cuvette at room
temperature in the dark. Check color reaction.
11. Dip slide 3x in PBS, pH 7.2.
Color reaction is terminated as soon as the pH is adjusted to neutral.
12. Hemalum nuclear staining:
Filter solution before use. Incubate slide for 1-2 minutes at room temperature, then carefully
for 10 minutes under running cold tap water; apply one final rinse in distilled water.
This stains the nucleus of all cells in the tissue and does not target only specific cells. Cell
nuclei stained this way appear blue under a light microscope.
13. Cover sections with Royalmount, 1-3 drops per section. Tissue must still be humid.
14. Polymerization:
Put slide on 60°C heating plate for at least 30 minutes.
15. Mount cover slip:
Dip slide in xylene (hood), add 1-3 drops of DPX Mountant, dip in xylene again, slip slide on
tissue and let dry.
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Results [10,11]
Overview of the results with cross sections of porcine jejunum. All pictures were taken at 50x
magnification and oriented the same way to allow for easy comparison. The staining results
are presented and evaluated separately in the following part.
CIV22 – Mouse IgG
C-3D5 – Mouse IgG
HM19-2 – Rat IgM
KG 7-30 – Mouse IgG
LU-5-PM – Mouse IgG
PB1 – Mouse IgM
PE1 – Mouse IgG
PG1 – Mouse IgM
PM1 – Mouse IgG
PM-2K – Mouse IgG
PP1 – Mouse IgG
PP2 - Mouse IgG
Figure 6 Overview of the staining with the antibodies used in this paper (magnification 50x).
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Antibodies
C IV 22[24]: anti Collagen IV
Figure 7
Figure 7: The antibody has stained the muscularis externa and the muscularis mucosae as
well as cells in the epithelial layer of the villi. The submucosae has not been stained, neither
have any cells in the lumen. (dilution 1:200, magnification 50x)
Figure 8 & 9: Detail showing of the intestinal villi. (dilution 1:200, magnification 125x – left,
500x – right). The antibody was diluted in the concentrations 1:100, 1:200 and 1:400. The
1:100 and 1:200 yielded optimal results.
Figure 8 Figure 9
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C-3D5[25]: anti Trefoil Factor
Figure 10
Figure 10:. The antibody has stained the muscularis externa and the muscularis mucosae
as well as the villi and the endothelium of the blood vessels14. Cells in the lumen have also
been stained. The submucosa has not been stained. (dilution 1:200, magnification 50x)
Figure 11&12: Detail of muscularis externa, the submucosa, the muscularis mucosae and
the villi from left to right. (dilution 1:200, magnification 125x – left, 500x – right). The
antibody was diluted in the concentrations 1:100, 1:200 and 1:400. The dilution of 1:200
yielded the best results.
Figure 11 Figure 12
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HM 19/2[40]: anti Smooth Muscle
Figure 13
Figure 13 The antibody has stained all tissue. The submucosa is stained slightly lighter than
the rest. The border between villi and lumen cannot be seen. (dilution 1:50, magnification
50x)
Figure 14: Detail shot shows that the antibody has reacted stronger with smooth muscles
to the left and the muscularis mucosae than with the submucosa. (dilution 1:50,
magnification 125x)
No higher magnification shots were taken. The antibody was diluted in the concentrations
1:25 and 1:50. Both dilutions showed oversaturated results. This was the only antibody from
a rat host.
Figure 14
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KG 7/30 [26]: anti Endothelial Cells
Figure 15
Figure 15: The antibody has stained the blood vessels of the villi, the submucosa and in
between the muscles. (dilution 1:40, magnification 50x)
Figure 16: Detail of the intestinal villi (dilution 1:40, magnification 125x)
Figure 17: Blood vessels along the border of the villus were stained. (dilution 1:20,
magnification 500x). The antibody was diluted in the concentrations 1:20, 1:40 and 1:80.
The dilution of 1:20 delivered the best results.
Figure 16 Figure 17
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Lu-5 [27]: anti Cytokeratin
Figure 18
Figure 18:. Staining occurred at the edges of the villi. The rest of the tissue shows no
staining. (dilution 1:500, magnification 50x)
Figure 19: Detailed picture of the villi and lumen. (dilution 1:500, magnification 125x)
Figure 20: A clear border at the villi can be seen. (dilution 1:500, magnification 500x). The
antibody was diluted in the concentrations 1:250, 1:500 and 1:1000. The dilution of 1:500
yielded the best results.
Figure 19 Figure 20
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PB1 [28]: anti Epithelial Basement
Figure 21
Figure 21: PB-1 seems to show no reaction with the tissue at this level of detail. (dilution
1:200, Magnification 50x)
Figure 22: On the left side a continuous staining along the epithelia of the muscularis
mucosae is visible, whereas staining along the epithelia of the villi is discontinuous. No
staining occurred in muscularis externa or submucosa. (dilution 1:200, magnification 125x)
Figure 23: Artefact which reacted strongly with the antibody, though it has nothing in
common with the swine tissue being examined. (dilution 1:200, magnification 500x). The
antibody was diluted in the concentrations 1:100, 1:200 and 1:400. All dilutions yielded only
very little staining.
Figure 22 Figure 23
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PE1 [29]: anti Endothelial Cells
Figure 24
Figure 24: PE1 shows staining reactions along the epithelia of the villi. Staining also occurs
at the endothelia of the blood vessels and scattered staining in the blood vessel. Stained
cells can be found in between the muscle layers of the muscularis externa and in the
submucosa. No positive reactions are found in the muscle tissue. (dilution 1:25,
magnification 50x)
Figure 25: Detail pictures of the intestinal villi. Clear staining along the epithelial of the
larger villi and scattered staining near the smaller villi. (dilution 1:25, magnification 125x)
Figure 26: Higher detail picture of the villi. (dilution 1:25, magnification 500x). The
antibody was diluted in the concentrations 1:25, 1:50 and 1:100. The dilution of 1:25 yielded
the best results.
Figure 25 Figure 26
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PG1[41]: anti Goblet Cells
Figure 27
Figure 27: PG1 has stained all muscle tissue, including muscularis externa, the muscularis
mucosae. Staining also occurred in the blood vessel. Epithelial layers of the villi are stained in
a darker red. Submucosa shows no reaction with the antibody. (dilution 1:25, magnification
50x)
Figure 28: Detail of villi. (dilution 1:50, magnification 125x)
Figure 29: Detail shots of the intestinal villi a clear border along the stained area can be
seen. (dilution 1:100, magnification 500x). The antibody was diluted in the concentrations
1:25, 1:50 and 1:100. The dilution of 1:25 yielded the best results.
Figure 28 Figure 29
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PM1 [30]: anti Neutrophils
Figure 30
Figure 30: This antibody shows scattered staining in the intestinal villi. More concentrated
staining is found around or in blood vessels. Neither any muscle tissue nor the submucosa
reacted with the antibody. (dilution 1:200)
Figure 31: Detail of the scattered staining within the villi. (dilution 1:200, magnification
125x)
Figure 32: Detail of scattered staining in villi. (dilution 1:200, magnification 125x). The
antibody was diluted in the concentrations 1:100, 1:200 and 1:400. All dilutions yielded good
results.
Figure 31 Figure 32
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PM-2K [31]:anti Macrophage
Figure 33
Figure 33: PM 2K has stained isolated cells throughout the submucosa and in the villi in the
immediate environment of the submucosa. Villi located further away of the membrane show
no positive reaction. Muscle tissue has not reacted with the antibody. (dilution 1:2000,
magnification 50x)
Figure 34: Stained cells in the membrane and in the immediate intestinal villi. (dilution
1:1000,magnification 125x)
Figure 35: Stained cells within the submucosa. (dilution 1:1000, magnification 500x). The
antibody was diluted in the concentrations 1:500, 1:1000 and 1:2000. All dilutions yielded
good results.
Figure 34 Figure 35
28
PP1 [32]: anti Thrombocytes and Endothelial Cells
Figure 36
Figure 36: Staining appears in the epithelial layer of the villi and in the endothelial layer of
the blood vessels. Some cells in between the muscle layers are stained as well. Furthermore
loose cells inside the blood vessel appear positive. No cells of the muscle or submucosa show
a positive reaction. (dilution 1:200, magnification 50x)
Figure 37: Detail of the intestinal villi.(dilution 1:200, magnification 500x). The antibody
was diluted in the concentrations 1:100, 1:200 and 1:400. Tissues treated with the 1:200
dilution yielded the best results.
Figure 37
29
PP2 [33]: anti Thrombocytes and Endothelial Cells
Figure 38
Figure 38: Staining appears in the epithelial layer of the villi and in the endothelial layer of
the blood vessels. Some cells in between the muscle layers are stained as well. Furthermore
loose cells inside the blood vessel appear positive. No cells of the muscle or submucosa show
a positive reaction. (dilution 1:100, magnification 50x)
Figure 39: Detail shot of the intestinal villi.(dilution 1:100, magnification 125x)
Figure 40: Cells inside the blood vessels have been stained as well. (dilution 1:200,
magnification 500x) The antibody was diluted in the concentrations 1:100, 1:200 and 1:400.
The 1:100 dilution yielded good results on all tissues.
Figure 39 Figure 40
30
PBS Test
Figure 41
Figure 41: This tissue has not been treated with any antibodies. It was incubated only with
PBS to ensure that all staining reactions seen in the other pictures originated specifically
from the applied primary antibody. The blue color is the result of the hemalum staining so
that the tissue can be seen.
31
Discussion
All of the tested antibodies developed for porcine tissue have reacted with the porcine tissue.
Also, all of the selected antibodies developed for human tissue staining showed positive
staining reactions. This is due to the epitope having the same or very similar chemical
composition in both species. It is therefore recognized comparably in porcine as in human
tissues.
The antibodies C-IV-22, C-3D5, Lu-5 and PG1, had an overall stronger color reaction with the
tissue, staining larger areas. Due to the more widespread distribution of their respective
antigen. The HM 19/2 antibody reacted too strongly with the tissue, leaving little or no
possibility to identify the antigen it actually binds to. Further tests with this antibody should
be done at higher dilutions. The other antibodies yielded good to intensive staining as well,
illustrated nicely by the pictures at higher magnification. However, the cell populations seen
are smaller, demonstrating their specialized function where needed. Also, some antigens are
expressed more frequently than others, which directly affects the intensity of the staining.
C-IV-22 [15,25]
This antibody binds to an antigen found on collagen IV. Collagen IV is found in basal lamina,
an epithelial layer that is secreted by the basement membrane. Through a light microscope
the basal lamina cannot be seen but is an indicator to where the basement membrane lies.
The C IV 22 antibody reacts nicely with the swine tissue. By staining the basement layer, it is
now possible to see the villi clearly.
C-3D5 [25]
The C-3D5 Antibody is known for specific binding to the Trefoil Factor 1 (TFF1) protein. TFF1
appears in the mucous gel covering the gastrointestinal mucosa. Since the mucous gel is no
solid tissue, but rather fills the inside of the gastrointestinal tract, staining also occurred in
the lumen. The two muscle layers of the muscularis externa are distinguishable, also the
blood vessels in the submucosa and the intestinal fold can be seen well. The intestinal villi
are hard to make out since the surrounding has been stained as well.
HM 19/2
HM 19/2, being the only tested antibody from a rat host, shows intense staining, even in the
tissue treated with a higher dilution of the antibody. HM 19/2 typically binds to an antigen
from smooth muscle cells. Though the muscles have been stained, a lot of tissue containing
no muscle has been stained as well. The submucosa and the blood vessels in it can be
recognized, as well as the border between the circular and longitudinal muscle. Transition
from villi to the lumen cannot be seen. Higher dilutions need to be tested for this antibody.
32
KG 7-30 [26]
KG 7/30 reacts positive with porcine endothelial cells, thus staining the vascular endothelial
cells in the blood vessels. This can be seen in the larger blood vessels of the submucosa but
also in the blood vessels of the villi. Interestingly, the higher magnification pictures (125x
and 500x) show that blood vessels are arranged along the border of the villi.
Lu-5 [16,27]
Lu-5 stains an epitope located on the surface of epithelial cytokeratin filaments. Cytokeratin
is a structural protein found in some cells. On the tissue, the epithelial layer which is in direct
contact with the lumen has been stained. Since the microvilli are where the epithelial layer
is, it is most likely that cells of the microvilli have been stained.
PB1 [28]
PB1 has only reacted with very few cells, so that staining can only be seen at higher
magnification. The antibody recognizes the epithelial basement cell layer. Where staining can
be seen, it has indeed stained the epithelial layer. It’s interesting to see that the cells stained
by this specific antibody are quite unique and that the layer they define is often hardly
thicker than a couple of cells.
PE1 [29]
This antibody marks blood platelets and endothelial cells of the blood vessels. The staining of
the blood vessels in the villi make this tissue section look very similar to the one stained with
the KG 7/30 antibody. However, the staining is fainter and no blood vessels in the muscularis
externa were stained, indicating that the two antibodies recognize different proteins.
PG1 []
The antibody PG1 marks porcine goblet cells. These are glandular epithelial cells whose
function is to secrete gel forming components of mucus. Since the epithelial layer of the
mucosa has a higher density of these cells, it has been stained more intensely. Some type of
goblet cell seems to be present in the muscularis externa since it has also been stained. The
complete structure of the gastrointestinal wall can be seen very well in this section.
PM1 [22,30]
PM1 targets neutrophils, a particular type of white blood cells. Neutrophils are the most
common type of white blood cells and belong to our innate immune system. Most of the time
they circulate in our blood stream, but they have the ability to migrate actively into the
tissue to destroy hostile microorganisms when needed. It is clearly apparent that neutrophils
have been marked in the blood vessels, but cells in the mucosa have been marked as well.
These are therefore neutrophils which have migrated into the mucosa to combat bacteria
and other microorganisms that are present there.
33
PM-2K [31]
PM-2K stains macrophages in most tissue. Macrophages (Greek: big eaters, from makros
"large" + phagein "eat"), are a type of white blood cell that engulf and digest cellular debris,
foreign substances, microbes, and cancer cells in a process called phagocytosis. In this tissue
section the macrophages are spread out evenly throughout the submucosa. It is noteworthy
that the macrophages stained by this antibody are mainly or exclusively located in the base
of the villi.
PP1 [32]
PP1 binds to platelets and endothelial cells. Much like the other antibodies (i.e. KG 7-30,PE1
or PP2) that have stained endothelial cells, the blood vessel endothelia has been stained.
This gives it the distinctive staining pattern near the outside of the villi. Platelets circulate in
the blood and can therefore be seen in the blood vessels. Platelets stained in other parts of
the tissue are hard to differentiate since stained blood vessels appear there as well.
PP2 [33]
The staining pattern of this antibody is very similar to the one of PP1 since both of them bind
to antigens located on the same type of tissue. The stained endothelial cells of the blood
vessels show once again the microanatomy of the villi and where blood vessels run through
the submucosa.
The antibodies have brought forth a nice result showing a broad spectrum of different cells
that were marked. Some of them staining larger areas of the tissue. Others marked only
isolated cells that seem to have specialized functions in our body. Only through the
immunohistochemical staining specifically of these cells were we able to differentiate the
tissues and fully recognize the anatomy of the intestinal wall. It was satisfying to see all of
the antibodies reacting with the tissue chosen.
34
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36
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