Week Seven

This week was quite busy and I was able to perform many new procedures, including real-time PCR and immunohistochemistry. Both processes were similar to one I have done before, standard PCR and Western Blotting, respectively. I have also become aware that, just like cells need care even when it's inconvenient, sometimes procedures will take you straight through when you wanted to eat lunch or late into the day and you have to figure out how to handle that. It usually it not as hard if you are busy - you can easily pass hours in what feels like minutes in a lab, especially if there are no clocks around you. However, working like that for hours straight - especially without eating much - can be tiring, so it is important to try to complete your most crucial tasks when you are most awake, in the morning or early afternoon, and to try to take breaks when you can. There are waiting periods in many procedures, and these are great chances to go get a drink of water or to eat a snack - or, if you're really tired, to go get some coffee. It is also good to keep in mind how long a certain procedure takes before you start it, so you know what you're getting into and if you are choosing a good time to start it with whatever else you have planned out for your day or week.

Real Time PCR is a method that is often used for analysis of RNA samples, since standard PCR does not really work with RNA itself, and doing the PCR analysis in real time instead of with a gel afterwards provides quantitative data that can be later manipulated with statistics - again, another use of Excel. Since the starting material is RNA, it must first be converted to cDNA through a reverse transcription process before it can be used for real time PCR. Real time PCR utilizes a machine that can monitor the polymerase chain reaction as it happens by noting when it sees the samples fluorece as the cDNA has progressed through another cycle. For our machine we must use 384 well plates, which have very small wells and can be a bit disorienting at first. Thankfully, our lab has a few multichannel pipettes which makes it so we can load multiple sample at once. Real time PCR seemed like a daunting task to me before I had tried it, but once I had I realized it was not as difficult as I thought it would be. The advice that the graduate student in my lab gave me was that the hardest part is simply remembering your place on the plate, and I feel like that is true for most procedures where you have to load samples, though it is a bit harder since there are so many more wells for this than ever would be for a standard PCR or a Western Blot.

Figure 1. 384 well plate with labels (E&K Scientific).

Figure 2.  Multichannel pipettes (Labnet).

Immunohistochemistry is one of those lab procedures that not only can provide important information about the morphology of cells, but is also quite fascinating and reminds me how science can allow us to look at parts of our world that we normally cannot. During many of the Friday lunch lectures that the mentors have been giving, they have been excitedly showing us pictures of cells that have been stained and imaged - even people who have their Ph.D.s already and have been looking at cells for years are still amazed by the beauty of these pictures. There are many different dyes that can be used for immunohistochemistry, and the the fluorescent dyes give some of the most interesting pictures. Two are shown in below: the DAPI fluorescent dye stains the nuclei of cells, whereas phalloidin 448 stains the actin and the cytoskeleton (ThermoFisher).

Figure 3. Example of immunohistochemistry on HEK 293 cells, using
fluorescent dyes DAPI, blue, and phalloidin 448, green (Bruce, 2013).

The protocol for immunohistochemistry was explained to me initially by the technician I work with in regards to its is similarity to Western Blotting, since it requires the use of a primary and secondary antibody. The overall procedure combines many different techniques, as first cells must be grown and treated appropriately, then cover slips have to be probed so they can imaged under a microscope that is attached to a computer that allows pictures to be taken and distributed.

Figure 4. A schematic of how the primary and secondary* antibody allow
visualization of the desired proteins (Leinco Technologies, Inc).
*secondary antibody is labeled here as "fluorescent tag," which is also accurate

I've again been having issues, this time with my cells. A few weeks ago, I had frozen down cells to store them and have been plating them for my project. The first time I plated them everything went fine, and I continued to grow them for a bit. One of the later times when I was splitting my cells, I believe that I did not do it aggressively enough and when I checked the dishes later under the microscope, I looked around the entire plate and could find no cells. I had been leaving plates in the incubator with only media in them as I must have left the cells in the plate I discarded when trying to split them. However, I was not incredibly concerned because I still had more aliquots of my frozen down cells, and I simply plated one of those. When I checked those cells, they were absolutely dead. I tried plating more cells, and they were also dead. Next week I will be attempting to plate cells again, using aliquots that someone else had froze down in case an issue occurred when I froze the cells down. Something could also be going wrong as I am plating the cells, but overall I just hope that the cells will adhere to the bottom of the plate and grow as they should, as I need them to complete the next tasks for my project.

References:

Bruce, D. 2013. Cell culture and stem cell techniques. <http://www.laney.edu/wp/doug_bruce/cell_culture_stem_cell_training/>.

E&K Scientific. 384 Well Roche 480 Plate, White, Barcoded. <http://www.eandkscientific.com/384-Well-PCR-Plate-White-for-Roche-Light-Cycler-Barcoded.html>.

Labnet. BioPette Plus Autoclavable Multichannel Pipettes. <http://northamerica.labnetinternational.com/products/biopette-plus-autoclavable-multichannel-pipettes>.

Leinco Technologies, Inc. Immunohistochemistry Protocol for Frozen Sections. <http://www.leinco.com/immunohistochemistry>.

ThermoFisher. Alexa Fluor 488 Phalloidin. <https://www.thermofisher.com/order/catalog/product/A12379>.