Wednesday, May 18, 2011

Our Accomplishments

Histochemical Staining

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.


Fluorescence Staining

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.

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