Day No. 5!

ITS FRIDAY!! TGIF!

We started the day with observing the stem cells we cultured yesterday. They changed from spherical to spindle-shaped. Also all of them are aligned in the same direction as the stamp we used yesterday. It was pretty cool. Too bad we don't have pictures. :(

So now we will be starting with the immunostaining procedure!

1. After removing the culture medium, the cover slips were washed with PBS using the shaker.
2. parafomaldehyde is added for cell fixation, which is to kill the cells while keeping the cells' original condition. If left for more than 10 minutes, it could de-sensitise the antigens present in cell. Because parafomaldehyde is light-sensitive, we had to work in semi-darkness.
3. Adding triton (not Neptune's moon nor the Greek god) to the cells would open up pores in the cell surface membrane, allowing antibodies to enter the cell. 
4. BSA is added and it binds to the unwanted parts of the cell. This ensures that the antibodies would bind to the antigens only, and not any other particles in the cell. This is possible due to the difference in affinity level.  To ensure that BSA solution stays on the cover slips and to increase effectiveness, the cover slips were transferred onto hydrophobic Parafilm. Because of its hydrophobic nature, the BSA solution would be repelled and remain on the cover slips.
5. A specific antibody is added, which will bind to actin in cells. The antibody carries a red dye, which is used to identify the organelles when placed under the fluorescent microscope. Another antibody, that would bind to the nucleus instead, was added afterwards. It carries a different-coloured dye. The anitbodies are light-sensitive, so we also had to do in semi-darkness.
6. We used the fluorescent microscope to see the nucleus and actin. IT WAS REALLY COOL.

There are a lot of different antibodies we can use to identify different organelles in the cell. However, due to time constraint, we are only limited to the nucleus and actin. The antibody used must be specific to each organelle, in order to identify the organelles accurately. Different antibodies take a different amount of time to bind. For example, the anitbody for actin takes about an hour, while the antibody for the nucleus only takes 10mins.

A/P Tan dropped by to have a chat with us. She gave us a summary of what we went through in the past week, starting from electrospinning to grafting. She explained how tissue engineering is an alternative to organ transplant and went through the pros and cons of it. Similar with organ transplant, tissue engineering also has its own risks such as rejection, especially when the cells are taken from another donor. Drugs may suppress the immune response, but result in opportunistic infections. But tissue engineering is a faster method than organ transplants, as the waiting time may be shorter.

We will be back next week in a new school, School of Biological Sciences! Ivan will be our next mentor and he is REALLY TALL. We hope that we can learn something new to add on to our Biology knowledge! Have a happy weekend!

Working in semi-darkness.

It's dark in there. BOO!

Measuring BSA powder, using a very sensitive electronic weighing balance. The powder is dissolved in PBS to make the BSA solution.

The fluorescent microscope. A variety of different coloured lights can be used, such as red and green. Different staining require different light colours. For the nucleus, we used red light, while we used green light for the actin. Oddly enough, the nucleus were seen in blue and the actin in red. BIZARRE.

Microscope pictures of actin. REALLY COOL.

The blue spots are nucleus.

We had a great time with Ajay over the past three days! Thank you, Ajay!