Goh Jun Hong - S209
Daniel Pei Zheng De - S208
Abu Ubaidah - S206
This project primarily concentrates on the histology and morphology of cells and for us (the students) to learn and appreciate the usefulness of the light and fluorescence microscope. The project is focused on the histological preparation of tissues. We will then be introduced to the 3 type of histological preparation :
a) Histochemical staining
b) Immunohistochemical staining
c) Fluorescence dye staining
With reference from Nayang Technological University: SST-NTU Flagship Programme: Project 2: Project 2 Manual.pdf
This project intends to achieve a deeper understanding on liver cells and how cancer cells may affect them in terms of behavior through the use of staining cells through the use of technology, Namely using immunohistochemistry, histochemistry and fluorescence staining methods.
- Histochemical Staining
1) What are the colours of the stained tissue?
Pale pink and Purplish-pink.
2) What is the pattern of colour distribution in the stained tissue?
There are about 25% Purplish-pink and the rest are Pale pink.
3) Can you identify which are the nuclei of the cells? What colour are they?
Purplish-pink round spots are the nuclei of the cells.
4) Can you identify the cytoplasm of the cells? What colour are they?
The pale pink Y-shapes are the cytoplasm of the cells.
5) What are the relative sizes and shapes of the cells in the tissue?
6) How many different types of cells do you think there are in the tissue?
1 type of cell.
7) Are there any other observations you identified? If so, describe them.
The dye was unevenly spread thus could contribute to an unreliable result.
- Immunohistochemical Staining
1) What are the colours in the tissue?
Light brown and a tinge of green.
2) What is the pattern of colour distribution in the stained tissue?
10% brown surrounding the 90% green.
3) Are the colours and pattern of colour distribution similar to that in the first slide?
4) The purpose was to determine the presence and location/distribution of a protein called vimentin. Which colour do you think represents vimentin? Why?
Light brown. Cells produce vimentin thus the surface area of the cell should be surrounded by vimentin and light brown was observed on the surface around the cells.
5) Describe the location/distribution of vimentin in the tissue.
It is distributed unevenly and found in the greatest concentration around the cells.
6) What does the other colour(s) represent?
It represents the cytoplasm of the cells.
7) Are there any other observations you have identified? If so, describe them.
- Fluorescence Staining
1) Can you see the tissue using the fluorescence microscope? Does the tissue apear similar to the first and second slide?
The tissue is not visible under the fluorescence microscope and the organelles observed are nothing similar to the first and second slides.
2) Describe what you observe. How many colours appear when using the fluorescence microscope and what are the colours?
Blue and Black colours were observed.
3) What do you think the colours represent? Why?
Blue represents the nuclei of the liver cells while black represents the background and the rest of the organelles of the cells that cannot be stained by the Hoescht dye. The hoescht dye becomes visible as a blue colour under the UV lights at a certain wavelength and since it only attaches to the DNA in the nuclei of the liver cells, the nuclei appears to be blue while the unstained portions of the tissue appears black as UV lights are not visible naturally.
4) Can you determine what is/are the pattern of location/distribution of the colours in the tissue? If no, why not?
No, we do not know how the cells in the liver tissue looks like under the fluorescence microscope as it only shows us the nuclei of the cells.
5) Are there any other observations you have identified? If so, describe them.
Yes, all the nuclei of the liver tissue appears to be of the same shape, This can attribute to identifying different substances quickly.
With reference from Manual for applied challenge project, Dr. Kenneth Yu, Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University.
Histochemistry is the branch of science concerning tissues and its identification and distribution by means of stains and indicators through chemical and microscopic means. What we did was basically stain colourless liver cells which are hard to view under a normal light with Eosin and Hematoxylin. Through this experiment, we managed to get more insight of the light microscope such as the magnification of the eye pieces, describe different parts of the cells such as the differentiation between the nucleus and the cytoplasm observed in cells after the staining as they appeared in different colours and we also managed to get first hand experience in handling tissues in preparation for histochemical staining. Finally, we also managed to observe different structures of the human liver samples and how they would appear different with cancer (i.e, for liver cells, they appear further apart and less ‘bonded’ as cancer makes the liver grow further with tumors and such.) The colours observed were dark blue for the nuclei, purple and pink for the cytoplasm.
Immunohistochemical Staining Protocol
It is a method to identify a specific protein in cells using a primary antibody that binds to that one particular protein, then a secondary antibody is bonded to the enzyme that targets the first antibody. The enzyme on the second antibody catalyzes a colourless solution to form a colored product, produced by the cells.
A primary antibody, that is targeted to a protein named vimentin, is given to us to be spread on to the liver tissue, and to be incubated for a full hour. It was placed into the buffered solution called PBS. Then a peroxidase-conjugated secondary antibody solution is applied and was left to incubate for 45 mins. It was then again place into PBS. Then an enzyme substrate solution was applied onto it, and was left to incubate for half an hour. Then it was placed in PBS again and rinsed in distilled water. A drop of mounting media is applied and dried for 5 mins. Then we looked into a light microscope to inspect our results.
Some parts of the liver tissue is stained in a light brown colour. If the light brown colour is significant in liver tissue, it means that much vimentin is present, thus it is more likely that cancer cells are present too, since cancer cells excessively produced vimentin. It was then concluded that our liver tissue is most probably infected with cancerous cells.
The goal of this technique of staining is to identify and locate organelles in a cell, namely the nucleus and the mitochondria. The dyes can only be viewed though the use of a fluorescence microscope. The microscope splits light into different wavelengths. Fluorescent dyes can only be seen when a specific range of wavelength hits them. Each dye has their specific range of wavelength which excites them to fluoresce and they give out photons which can be seen by us. The dyes used in fluorescence staining sticks to specific organelles like the nucleus or mitochondria. This happens because the dyes used are positively charged and thus, would be attracted to DNA which are negatively charged. DNA is found in the nucleus.
For this project, the objective was to stain the nucleus of liver cells in a slice of liver tissue and observe it under a fluorescence microscope. The dye used to stain the nucleus is the Hoescht dye, which emits the colour blue. The background of images is black. This is due to the UV light that is projected at the DNA of the nucleus/mitochondria of the liver tissue that is binded with the Hoescht dye and it reflects the UV light into our eyes to be seen, while the background is black due to the fact that the background does not have DNA and the Hoescht dye is not binded with it thats why the UV light could not be reflected into our eyes to be seen, thus we see black.
-Purpose of this method is to identify and locate organelles in a cell through the use of fluorescent dyes and a fluorescent microscope.
-The dyes are positively charged and the DNA, found in the nucleus, are negatively charged.
This project has indeed deepened our understanding in Microbiology, we learnt in detail on how to use light and fluorescent microscopes. We also learnt on how liver cancer could be diagnosed through immunohistochemical methods by detecting vimentin, which if present in high levels in the liver could attribute to cancer. and how nucleuses could be identified via UV lights emitted through a fluorescent microscope. UV lights can be used at crime scenes as well to determine if blood was indeed real at such a crime scene involving human white blood cells, which has a certain definite shape for its nuclei. Next, the histochemical staining methods gave us a better understanding on how to handle cells in preparation for staining with eosin or hematoxylin, this broadening our knowledge further to understand how to identify cancerous cells at early stages in the liver, a telltale sign being that the liver cells are further apart in the samples. In the future, we might teach others on how to conduct such experiments since we have had our experiences in handling such tissues already, given the facilities necessary.