Tissue Morphodynamics Laboratory: Weeks 4

Hi, this is Danny with a couple of updates regarding my progress in Dr. Nelson's Tissue Morphodynamics Lab. My next three posts are going to be a load of information, as I learned a lot of new material and methods over the course of past three weeks.  I understand that these posts are little late, but it could not be helped, as my laptop with all images crashed. I felt that the words I was writing would not do the  techniques or methods any justice so I figured I would wait for my laptop and my images. Just to note, these three posts are actually just one post, but I just prefer to have the weeks organized as there is going to be a lot information being posted.

My lab bench where I stain, aspirate and fix the lungs.

One of the three dissecting microscopes I use.  

The fourth week at my lab was definitely more exciting and productive than previous weeks, as there were no interruptions or unforeseen obstacles. In addition to a new high school student, Chris, from Montgomery High School joining the laboratory, there were also three four new senior thesis students and an undergraduate student. Needless to say the lab got crowded very quickly and I found myself changing desks numerous times. Although the dissecting microscopes were usually occupied during the mornings, I found myself with microdissecting chicken embryos the entire afternoon from mid-day to around five. My first day of microdissecting day six chicken embryos was somewhat embarrassing, as I microdissected ten or so chicken kidneys instead of the lungs. Having been told that the lungs are slightly larger in day six embryos than day five, I found myself with a plate of chicken kidneys. Although ten or so chicken embryo unfortunately were lost, I found that microdissecting chicken lungs was a lot easier than trying to take out the kidneys. Around the middle of the week I already had fifteen or so day 5, day 6, and day 7 chicken embryo lungs waiting to be fixed, stained, and imaged. 

Although I went into a little detail regarding the staining of the epithelium in my previous post, I figured that I would go into a little bit more detail, as I see other EXP students are staining as well.

Throughout the microdissections and the staining procedures, Phosphate Buffered Saline (PBS) is crucial in order to preserve the lungs from an hour up to months. After successfully dissecting at least six chicken embryo lungs I head over to my lab bench with my dish of lungs submerged in PBS and start to fix the lungs. In order to make the formaldehyde solution for fixation, I add 10mL of stock 16% paraformaldehyde (PFA) solution to 30mL of 1x PBS, resulting in a 4% PFA|PBS solution. Then I add the lungs into a well, usually three or four to a well, and then aspirate the excess PBS solution using an aspirator and add the 4% PFA|PBS solution to the culture dish. I then place the dish on a shaker for amount 15 minutes and then aspirate the PFA|PBS solution and washing the excess PFA|PBS solution three times with PBS. Next, I prepare a Phosphate buffered Saline with Triton X solution (PBST) by adding 1.5mL of Triton X into 500mL of PBS, and add a stir bar and leave it on a stir plate for about 30 minutes in order to make a 0.3% PBST solution. Triton X is a very viscous solution and is used in order to make holes in the tissue and mescheyme, which allows for antibodies to react with the epitopes for primary binding in staining. To continue fixing the lungs I add the 0.3% PBST solution to lungs for about 15 mintues on a shaker. Then I add a blocking buffer, which consists of 4mL of 10% goat serum (GS) and 30mL of 0.3 PBST, in order reduce background noise when staining and imaging. I leave the lungs submerged in in the blocking buffer in 4°C for four hours on the shaker, as GS tends to attract bacteria if left at room temperature. After four hours I add the primary antibody, which consists of 1mL of PBST and 5µL of LCAM. After four hours and aspirating the blocking buffer, I add the LCAM primary antibodies overnight at 4°C on the shaker. When I return the next day, I aspirate the LCAM solution and then wash the lungs in PBST for about 4 hours, aspirator and adding sterile PBST every hour. Then I add the secondary antibody, which consists of 488 nano-meter (turquoise color) goat-a-mouse serum. I wrap the plate of wells in tinfoil and leave it overnight. When I return I usually spend up to two days constantly aspirating and adding new PBST to the well of lungs, in order to prepare for imaging. As one can see staining takes about half week at a time, so it is best to have many lungs to stain at once than repeat the process multiple times. 

Although the fourth week mainly consisted of microdissecting, messing up microdissections, and then staining the lungs in order to prepare for imaging the following week. I also learned a lot more about the work others were doing in lab through the group meetings, which consisted of Oscillatory Rotation and Cell Motility and the Mechanics in Meschyme-free branching. What was interesting in the latter topic was that the post-doc cut the distal tips of chicken embryos, which reportedly are places of high stress and proliferation, and embed them in matrigel which was submerged in fibroblast growth factor (FGF), a family of growth factors that are key in embryo develop. Using time lapse imaging on the confocal microscope, he was able to create a animation of the terminal end buds actually growing out of these square matrigels in culture. Although similar works has been done, which I will provide an image in a second, I don't believe anyone has necessary done the work recently using modern technology. 

An  general idea of the project discussed during the group meeting.
Source: Fibroblast Growth Factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung

Lastly, in between staining and microdissecting chicken lungs, I was busy learning other useful techinques that may or may be useful in the near future. I learned the process behind making complementary DNA (cDNA) and quantifiable polymerase chain reactions (qPCR), which is similar to the polymerase chain reactions (PCR) we did in AP biology. In short, the entire cDNA process consisted of following a instruction booklet sent from Thermoscientific Fisher and numerous attempts to find a optimal amounts of solutions to add. qPCR was similar to PCR except that the procedure would allow for one to quantify the relative amounts of increase in DNA overtime relative to other samples using spectrometer, whereas in PCR one could only analyze the amounts of DNA after running the DNA through gels and imaging them in ultraviolet light. 

The fourth week was a lot of new and exciting research, as well as new and exciting methods and techniques. I learned a lot during the fourth week and got to know a lot of the people in lab, besides Amira and Ben.