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This experiment was performed to identify microtubules in Nb2a cells through immune staining using α tubulin antibodies and enzymatic reaction. In this experiment Nb2a cells were fixed using the 4% paraformaldehyde. Cells were permeabilized with the 0.2% Triton X-100. Cells were blocked using the 3% bovine serum albumin (BSA). Adding the primary antibody cells were incubated for the 45 min. Alkaline solution and BCIP/NBT added to the cells and cells were viewed under the microscope. In the result we detected the dark brown precipitate in the positive cells. This was the reaction of the alkaline phosphatase linked to the secondary antibody with the BCIP/NBT reagents. Most staining was in the cytoplasm and the extensions that protrude off the cell body.
Immunocytochemistry methods are used to identify the proteins and other molecules in the cells by using the specific antibody that bids to it and identifies the protein. In immune staining usually there two antibodies involved; primary and secondary. Primary antibody is targeted to an epitope of the protein of interest. The use of antibodies in the multipart and mixed environment of the cell at times gives unexpected binding not dependent on specific binding of the primary antibody to the correct antigen .
Immunocytochemistry started in 1942 when Albert coons and co worker reported using the fluorescence labeled antibody in the liver segment. Researcher stated using the control from the use of other antibodies. Primary antibody is useful to changes in level and changes in the phosphorylation. Secondary antibody binds to the primary antibody and they detect the proteins or other molecules. The secondary antibody is conjugated to a detection system that encodes an enzyme that yields to color product. It is an important tool for the resolve of cellular contents from different cells.
Material and Method:
Students will receive 3 plates with the Nb2a cells and one plate will be the negative control and students will perform immunocytochemistry on other two plates. First step is to fix the cells. To fix the cell remove all the media from the plates and slowly add the 4% paraformaldehyde and let the plates sit for 5 minutes at room temperature. After 5 minutes take out the paraformaldehyde and rinse the plate with the 1X PBS. Let the plate sit for couple of minute and remove the PBS. Second step is to permeabilize the cells add 1 ml of 0.2% triton X-100 and let the cell sit for 5 minutes at room temperature. Wash the cells with the PBS and let the plate sit for 2 minutes and take out the PBS. Third step is to block the cells by adding the block antigens by adding 1ml of 3% BSA in Trish buffered saline and let the plates sit for 30 minutes at room temperature. This blocking agent will block any non specific binding of the primary antibody to non tublin proteins. To bind the antibody add 1ml of primary antibody to each positive plates incubate the pates for 45 minutes at 37°C. Add 1 ml of blocking solution to only negative plate. After 45 minutes of incubation take out the primary antibody and transfer it back in to the original tube. Rinse the cells 3X with 1X PBS and each time incubate the plates 5 minutes at room temperature. When you rinsing the cells be careful with removing the media and adding the media to the plates. Add 1 ml of secondary antibody to both positive and negative control plates and incubate the plates for 45 minutes at 37°C. Secondary antibody is diluted in 3% BSA at 1:2000. Rinse the cells 3X with 1X PBS and incubate the plates for 5 minutes at room temperature. Once more rinse the cells with 1 ml of alkaline buffer. This alkaline buffer will help in maintaining the optimum pH for the conjugated enzyme alkaline phosphate. To observe the enzymatic reaction remove the alkaline solution and add 1 ml of substrate solution BCIP/NBT. Add this regent to the both positive and negative control plates. After the plates are observed by the T.A stop the reaction with by removing the substrate and adding the distilled water to the plates.
Fig 1: Negative Control plate Fig 2: Positive Control plate
Table 1: Cell confluency at staring and at the end after substrate solution is added.
Confluency at starting
Confluency at end
Positive control # 1
Positive control # 2
Table 2: Efficiency of stained cells showing the immunoreactivity to antibody.
Stained Cell #
Positive Control #1
Positive Control # 2
As you can see in the table 1 the confluency of the cell went down at the end of the experiment. Cells confluency went down because the cells were washed so many times and that time some cells might be pipette out from the plate by mistake. Fig1 shows that negative control didn't had that much immunoreactivity with the antibody. There were only couple of the cells were stained. Fig 2 shows that positive control had immune reactivity and most of the cells were stained blue. In Fig 2 you can see the cells are stained around the nucleus. Cells are most stained in the cytoplasm. Table 2 shows the efficiency of the cells that stained cells in both controls that had immunoreactivity with the antibody.
During this lab period we have learned that how the primary and secondary antibodies are targets the specific protein of interest and how it is bind to the protein. We also learned about how cells are fixed, permeabilize, blocked, and how they bind to specific antibody. Immunostaining is used to detect the distribution and localization of the specific protein within individual cells . The result for this lab was as expected. We expected that positive control plate cells will bind to the substrate solution and cells will be stained blue and negative control plate cells will not bind to the substrate. But we also found some cells stained in the negative control plate. Positive control plate's cells showed the innunoreactivity to the antibody and the cells were stained blue. Stained part of the cells were cytoplasm and outside the nucleus. Microtubules that form the cytoskeleton are made up of a protein called tubulin and are present abundantly in neuron. For this experiment primary antibody was anti tubulin. Secondary antibody provides the universal labeling and label amplification. The cell confluncy went low because by the end of the experiment I think cell started to die and some cells are stuck on the side of the plate. Both positive controls had immunoreactivity to the antibody and the negative control plate did not have immunoreactivity to the antibody.