Immunohistochemistry to study Human Xenografts for Expression of SMA and Endomucin

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23/09/19 Sciences Reference this

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The use of Immunohistochemistry to study Human Xenografts for Expression of SMA and Endomucin

Introduction

Immunohistochemistry (IHC) is an important which localizes antigens within a tissue section and exploits the ability of antibodies to bind strongly and specifically to these antigens. Monoclonal antibodies or polyclonal antibodies are used in IHC to determine the specific antigen distribution in a tissue (Kaliyappan et al., 2012). These different types of antibodies are created via different means with polyclonal antibodies produced in host animals to raise antibodies against a specific antigen while monoclonal antibodies are created by harvesting spleen cells from an immunised animal and fusing them to a tumour cell (hybridoma) after which they should proliferate indefinitely and then they would screen the clones for good antibody properties. IHC is widely used for scientific research, the diagnosis of patients and in drug development (Kaliyappan et al., 2012). The properties of antibodies are used in indirect immunohistochemistry, where many different labelled secondary antibodies can be layered onto the primary antibody-antigen complex which provides a stronger signal and a greater sensitivity to the targeted antigen.(Giorgadze et al., 2012)

In this experiment the Avidin-Biotin-Complex (ABC) method of IHC is used. Avidin structurally has four binding sites for biotin and the interaction between avidin and biotin is one of the strongest non-covalent interactions in the nature (Jain and Cheng, 2017). This indicates that these proteins bind strongly and with high specificity to biotin. The ABC method for antigen detection exploits the properties of avidin by using antibodies conjugated to biotin. A biotin molecule is conjugated to antibodies and enzymes, and in the ABC-method the secondary antibodies are conjugated to biotin. The secondary antibodies are known to be biotinylated. When conjugated to the antibodies, biotin functions as a bridge between the tissue-bound primary antibodies and the avidin-biotin-peroxidase complex (Key, 2009). In this way, the signal from the primary antibody is amplified and this is shown in Figure 1.

 

 

Figure 1: A diagram to represent the ABC staining method. 

Endomucin is a sialomucin that can be found on the surface of endothelial cells in different animals including mice and humans (Kuhn et al., 2002). It is a membrane glycoprotein that has a function in preventing cell adhesion by having a rod-like structure that extends to the cell surface (Samulowitz et al., 2002). Human endomucin is expressed in highly vascular areas of the body like the lungs, heart and kidneys (Liu et al., 2001). Within the structure of the heart the human endomucin is expressed in aortic endothelial cells (Liu et al., 2001) unlike mouse endomucin which is only expressed in the capillaries and venules of the circulatory system (Morgan et al., 1999).

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In vertebrates six different isoforms of actin exist (Clement et al., 2007) and in this experiment the -smooth muscle actin (-SMA) is the focus of one experiments. Actin plays an important role in the body via control of cell motility and morphology that is dictated by microfilaments in which actin is an essential component (Clement et al., 2007). -SMA is only present in smooth muscle cells and is therefore used as a marker for this tissue type (Skalli et al., 1986).

Methods and Materials

Four paraffin-embedded sections of a human xenograft grown in a mouse were provided. We will stain for alpha-smooth muscle actin (Mouse anti-SMA; Santa Cruz Laboratories: SC-65495) and endomucin (rat anti- endomucin; CODE V.7C7.1). We will use Vectastain ABC Kit (Vector Laboratories) reagents to perform the labelling as described below. In order to begin the experiment and stain the sections properly the paraffin wax needs to be removed and the tissue needs to be re-hydrated. This is done by dewaxing the slides using these steps:

  1. Xylene 5mins X2
  2. 100% Ethanol 2 minutes
  3. 400ml of Methonal(Me0H) / 6ml of 30% hydrogenperoxidase (H202) for 15 minutes (This step is to inhibit the activity of peroxidase enzyme that naturally occur in tissues)
  4. 100% Ethanol 2 minutes
  5. 80% Ethanol 2 minutes
  6. 70% Ethanol 2 minutes
  7. 50% Ethanol 2 minutes
  8. Distilled Water 2 minutes

After this the citrate method is used as sometimes the paraffin embedding and dehydration process will distort or hide epitopes and this method can be used to ‘reveal’ the epitopes for the antibodies to bind to. The steps used to perform this are:

  1. Preheat citrate buffer (pH6) for 6 minutes in microwave
  2. Top up and put slides in the citrate buffer and microwave for another 10 minutes
  3. Slowly cool down the slides under running water
  4. Place the slides in the humid chamber and to ‘wash’ the slides with PBS (add PBS, incubate for 2 minutes, tap off PBS and then add PBS once again for another 2 mins)

The non-specific binding of antibodies to the slides must be blocked and there are 2 steps that occur to perform this function:

  1. Add blocking buffer (approximately 200ul per slide) on each of the slides for 10 minutes (NB: make sure to close the lid of the humid chamber)
  2. Wash the slides with phosphate blocking solution (PBS) BS 3x- pour off the blocking solution and add PBS to the section for 2 minutes, three times.

After all these steps have occurred each slide will then undergo a different treatment with 4 different steps done to each slide. These steps are outlined in Table 1. The sections were incubated with 200μl of the relevant primary antibody for thirty minutes, as shown in Table 1. Following washing with PBS 3 times for 2 minutes each time, the sections were incubated in 200μl of the appropriate secondary antibody for 30 minutes, as seen in Table 1. The slides were washed with the standard procedure using PBS 3 times for 2 minutes each. For the third layer, each sections underwent the same treatment with the addition of 200μl of the reagent for 30 minutes after which they were again washed with PBS 3 times for two minutes each time. The final layer is only used on the slide for 1-3 minutes after which washing in PBS occurs 3 times for 2 minutes. The slides are then counterstained with haematoxyin for 2 minutes and washed in tap water for 1-2 minutes. They are then dehydrated and mounted in DPX so that the staining pattern can be studied under a microscope. 

Table 1: The Different Layers Applied to Each Section

 

 

Section 1

(SMA)

Section 2

(Mouse control)

Section 3

(Endomucin)

Section 4

(Rat control)

1st layer

Primary antibody

Mouse monoclonal anti-SMA

Anti-mouse IgG

Rat monoclonal endomucin

Anti-rat IgG

2nd layer

Secondary antibody

Biotinylated anti-mouse IgG

Phosphate buffered solution (PBS)

Biotinylated anti-rat IgG

Phosphate buffered solution (PBS)

3rd layer

Avidin-Biotin-HRP complex (ABC)

Avidin-Biotin-HRP complex (ABC)

Avidin-Biotin-HRP complex (ABC)

Avidin-Biotin-HRP complex (ABC)

4th layer

Diaminobenzidene (DAB)

Diaminobenzidene (DAB)

Diaminobenzidene (DAB)

Diaminobenzidene (DAB)

 

Results

Heavy brown staining is observed only on the anti-SMA slides while there is only very light brown on the anti-endomucin slides. The control shows no brown staining. Each of the images has a clear background and no non-specific staining is observed. These observations are shown in Figure 2 below.

 

Endomucin surrounding a blood vessel

 

 

Hair follicle containing keratin

 

 

SMA surrounding a larger blood vessel

Figure 2: Slides of human xenograft grown in a mouse that has been subjected to immunohistochemical staining.

Discussion

Endomucin expression was only observed very slightly in the walls of the capillaries which enables us to see a clear area where high perfusion of molecules would take place. α-SMA is expressed in smooth muscle, therefore when testing for α-SMA, the brown staining was much more prominent due to the presence of smooth muscle.

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Staining was observed in both the endomucin and α-SMA experiments however the controls did not show any staining indicating that the tests were effective, and the expression was largely observed where expected. The considerable difference observed in the endomucin staining when compared to the α-SMA staining is due to the expression of α-SMA and endomucin being different in the skin from which the samples were taken. In order to confirm these results more repeats of the experiment can be done to ensure the pattern of expression.

In conclusion, immunohistochemisty is shown to be an accurate method to detect α-SMA and endomucin expression in the mouse skin sections which were grown from a human xenograft. This allowed for the identification of areas of smooth muscle and blood vessels in the sections.

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