Immunohistochemistry Staining

Pre-cut 10µm sections of formalin fixed paraffin embedded human brain tissue of HSVE and RTA controls were subjected for immuno-histochemical examination. The immuno-histochemical staining for CO1 and HSV-1 was performed on sequential slides. The slides were incubated with mouse monoclonal anti-CO1 (Abcam) and mouse monoclonal anti-HSV-1 antibody (Abcam), respectively. This anti-CO1 targets the CO1 protein that is embedded in the mitochondrial inner membrane. Details regarding the immuno-histochemical staining protocols were described in Chapter 2.

6.2.2 Quantitative Immunohistochemistry

The area of interest was randomly chosen for image analysis; however, it was necessary to avoid areas that comprised massive necrosis or haemorrhages. The images were captured from the same area from a consecutive slide that stained for CO1 and HSV-1 using a photomicroscope that was attached to the camera Nikon eclipse 80i (NIS-Element BR). The images were collected in TIFF format with a resolution of 1280 x 960 pixels and saved within the portable hard disk.

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The images were captured from the frontal, temporal, cingulate and amygdala regions of HSVE Patients 1, Patient 2, and RTA Controls 1 to 4. However, no images were captured from HSVE Patient 3 because of massive necrosis throughout the brain regions of this patient. No images were captured from RTA Control 5 because of weak staining from the slides of this case.

The image was captured under x20 magnification. Five images were photographed for each region and captured from the consecutive slides that were stained for CO1 and HSV-1. The analysis was performed using ImageJ.

First, the image was opened in the ImageJ program. Then, the background was substracted using the default factor that was set up by the program, which is 50. For an image with a dark background, the factor used varied from 10 to 50. A plugin for ImageJ called ImmunoRatio was then selected for the generation of pseudocolor images and the separation of positive and negative stained cells (Tuominen et al.

2010).

A pseudocolor/RGB image was split into three 8-bit images containing the red, green and blue components. The 8-bit blue image represented the positive stained cells and the red image represented the negative stained cells. These images were then thresholded. There was an automatic threshold that was provided by the program. The next step was the counting of the cells using a ‘particle tool’. A minimum value of 5µm was selected for the cell size settings. A value of 5µm was selected after measuring the diameter of the smallest nucleus in the image. Furthermore, this was supported by the fact that the size of the nucleus in most of cells averaged about 5µm. Finally, the outlines and summary for the number of

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counted cells was produced. The percentage of positive stained cells was calculated by dividing the number of positive cells with the sum total of the cells (negative + positive cells) and multiplying by 100.

Details regarding the quantitation steps were demonstrated in Figures 6.1 and 6.2. Figure 6.3 generally showed the proportion of the captured image under x20 relative to x4 magnification for the quantitation method. This image was provided to give a general idea of the size of the area involved in the quantitation.

150 Open ImageJ

Background subtraction

The factor varies between 10 and 50 Plugin ImmunoRatio for separating

positive and negative cell

Split Channel

Blue Red

Crop the region of interest

Threshold

Positive cell Negative cell

Threshold Analyse particle tool for

acquiring the cell counting

Analyse particle tool for acquiring the cell

counting

Figure 6.1 Flowcharts of the applications of the computer-based analysis of immunohistochemistry.

151 Step 4 Step 1 Step 5 Step 2 Step 3 Step 6 Step 7 Blue Green Red Blue

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Step 8 _{Step 9}

Step 10

Red

Figure 6.2 A flowchart outlining the analysis for particle cell counting. Step 1: An RGB original image was opened in ImageJ. Step 2: The first step of the analysis involved the background substraction. A default factor of 50 that was set up by the program was used for substraction. However, the factor varies between 10 and 50 for an image with a dark background. Step 3: An image analysed in the ImmunoRatio plugin. Step 4: An RGB pseudocolor image that was produced by Immunoratio was cropped, and the channel was split to produce three 8-bit images (red, blue and green). Step 5: The 8-bit blue image represents the positive stained cells. Step 6: The image was thresholded and the particle cells were counted. Step 7: The number of positive cells and the outlines of measured cells were provided at the end of the analysis. Step 8: The 8-bit red image represents the unstained cells. Step 9: The image was thresholded and the particle cells were counted. Step 10: The number of unstained cells and the outlines of measured cells were provided at the end of the analysis

153 A

B

Figure 6.3 The images A and B was captured exactly from the same part of hippocampus of HSVE Patient 1 but under different magnification. Magnification A = x4 and B = x20

154 6.3 Results

The results in this chapter are divided into two parts. The first part is the description for immuno-histochemical staining for CO1 and HSV-1, and the second part is quantification based on the computer analysis program.