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.