The focus of the project was to:
1) To introduce the basic concepts of how the light microscope works
2) Introduce the fluorescence microscope and how it works
3) Describe the characteristics of cells and how it relates to its functions
4) Describe and explain the functions of some organelles especially nucleus, mitochondria and endoplasmic reticulum
5) Introduce how tissues are prepared and have a hands-on experience on how it looks under the fluorescence and light microscope using 3 different techniques mainly histochemistry, immunohistochemistry and fluorescence dye techniques to stain the tissues and how we can view it under the various microscope.
Description of project:
In the project, we were involved in the preparation of slides for the observation of it using the light microscope and fluorescence microscope.The tissue samples were given to use beforehand. We used 3 methods namely: histochemistry, immunohistochemistry and fluorescence dye techniques to stain the tissues. First, we did histochemistry staining technique and observed the following:
where the dark purple areas represent the nuclei of the cell and where the pink areas represent the cytoplasm of the cells.
We then used the 2nd technique immunohistochemistry staining technique to stain the cancerous liver cells and observed the following:
where all the cells with vimentin are stained and where the dark brown areas represent nuclei of cancerous cells.
Finally, we then used the final technique while waiting for the slides to incubate for 45 mins. It was fluorescence staining. We then observed the following:
where the bright blue areas represent the nuclei of the cells.
1) During histochemistry staining technique, we observed that that the nucleus was stained purple while the cytoplasm was stained light pink. The cells looked compacted and seemed to be like a network and also consisted of a number of layers. The nucleus was stained purple yet the cytoplasm was not stained purple was due to the stain only staining the nucleus and not the cytoplasm. This concept also applies to the cytoplasm whereby the cytoplasm was stained light pink yet the nucleu was not stained light pink due to the stain only staining the cytoplasm and not the nucleus. This was done under 40x magnification under a light microscope.
Sketch of what we saw in the microscope:
2)While the immunohistochemistry slide was still incubating, we conducted the fluorescence staining. After staining the cancerous liver cells and placing the slide under the light microscope, we noticed that only the nucleus was stained white under fluorscence light and the background was black. This was so as the Hoescht Dye only targets the DNA found in the nucleus. There was a black background due to the ambient lightning so that we can see the cells under UV rays under the fluorscence microscope.
Sketch of what we saw in the microscope:
3) Finally we completed the immunohistochemistry staining. Under the microscope, we noticed that there were cells that were stained dark brown while other cells were stained light brown. This was so as the antibody(1) targets vimentin which was found in the cancerous liver cells. Then, we used antibody(2) which targets antibody(1) and left it to incubate. It also catalyses a colour-producing chemical reaction that enables us to see the brown colour in the photo. Finally, a substrate solution is added so that we can produce a coloured product that is precipitated on the exact location where the protein in question is found in the cells or tissue. We then viewed it under the light microscope.
Sketch of what we saw in the microscope:
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