Week 7 Mendelsohn Lab

This was my last week at the Mendelsohn lab. As I approached my last week, I felt it went by very quickly. I briefly learned how to use a digital microscope to take pictures of the slides that I have stained. Other than that, I did much of the same paraffin sectioning and staining, which felt interestingly comfortable.

One of the digital microscopes

On the last day, I bought doughnuts to share and thanked everyone for giving me the opportunity to work at the lab. I really enjoyed my seven week lab experience at the Mendelsohn lab and would highly recommend it to anyone who would be interested in working with cellular biology and genetics. Hopefully, I will be able to come back next summer.

This is the building where I worked

Week 6 Mendelsohn lab

Hi again. This is a summary of what I did during my sixth week at the Mendelsohn lab.

I briefly worked on Sol's silk bladder augmentation project by paraffin sectioning some of his blocks. I found these paraffin blocks much more difficult to cut because the silk embedded into the bladder tissue made it harder to fully slice without it ripping apart. 

Throughout my stay at the lab, I have been paraffin sectioning many mouse embryos, not fully understanding where and how these embryos have been embedded into the paraffin wax. During the week, Katya brought an E17 pregnant mouse into the lab to remove its embryos. She removed all 13 embryos from the mouse and placed them in 1x PBS, while I prepared 13 tubes of diluted formaldehyde fixing agent. Katya showed me the following steps to dissect a mouse embryo and told me to dissect the rest of the twelve embryos. First, I removed the amniotic sac and cut a small piece of its tail to be further genotyped through PCR. Then I bisected the embryo, under the arms, removing the upper half of the embryo (because we are only looking at the lower half). After bisecting, I moved under the microscope to clean out the rest of the embryo removing everything but the bladder and kidneys. After removing the existing limbs and tail, I placed the embryos into each of the 13 formaldehyde tubes to be fixed and eventually paraffin blocked at the histology department for future sectioning. 

Mouse embryo E17

                          

                                  Dissecting microscope

Under the microscope

The next day we ran a PCR on the small pieces of embryo tail (that I previously mentioned) and the following day we ran a gel to confirm the Cre genotyping of the embryos to see which had the gene and which didn't because eventually we want to cross mouses that have Cre with mouses that have Apaf mutations. 

Gel

If you have been reading, I haven't explained what this project has been really about. Basically, the Apaf project (the one involving mouse embryos) is about the connections between the ureter and the bladder. In embryos, the ureters are joined in the nephric duct through the common nephric duct. Normally, the ureters would detach from the nephric duct and fuse with the bladder epithelium. This project is trying to analyze Apaf (Apoptotic protease activating factor, one of the major proteins that form the apoptotic regulatory network) mutants to determine whether apoptosis is required for ureter insertion.

Week 5 Mendelsohn Lab

This is Jason again from the Mendelsohn Lab at Columbia. I have been behind writing my blogs and will update you all as soon as possible. I am writing this not actually in my fifth week, but here is what I did anyway.

Throughout the week, I have again been practicing and understanding how to stain slides using immunohistochemistry (briefly mentioned in my previous post). After paraffin sectioning and a day to let the tissue dry and settle on to the slide, these slides were ready for immunostaining. First these slides were deparaffinized in xylene solution and hydrated with ethanol so that the paraffin wax was fully dissolved, leaving only the desired tissue on the slides. After the deparaffinization process, the slides underwent heated antigen retrieval, which means the slides are placed back to back in a pH 9 buffer at 100˚C and steamed for 30 minutes. We did this because when the tissue is processed into paraffin blocks for sectioning, the tissue is added with fixatives that masks and cross-links its proteins, making successful antibody binding almost impossible. This way in the buffer and hot temperature, these fixed proteins were unfolded allowing our specific antibodies to successfully bind. After the 30 minute steam, the slides went straight into PBS .1% triton (a very common buffer solution) for 15 minutes to wash. Then horse serum blocking solution was applied to the slides for 90 minutes to reduce background or unspecific staining. After the blocking solution, the slides were ready for the specific antibody application. Lastly, I applied DAPI (a fluorescent DNA stain), washed in PBS .1% triton, and put on the cover slips. 

Staining hood (Deparaffinization on the right)

Slides deparaffinized and hydrated

Heated antigen retrieval

It might seem like a lot to remember at first, but after a couple times of practice you get the hang of it. 

My bench

On Wednesday, we had a formal lab meeting where everyone in the lab presented there work. I listened and learned as the other five lab members explained their projects. Katya, Kerry, Tammer, Hanbin, and Sol all amazed me with the work they were doing. The Mendelsohn lab focus spanned far beyond just bladder cancer and touched upon several different areas within the field of urology. For example Sol's project involves bladder augmentation using silk fibers as a scaffold to increase the size of the bladder and lower its pressure for impaired bladders.

I have continued my work on the BBN and Apaf projects and will explain them later in my next post. 

Weeks 3-4 Mendelsohn Lab

Hi this is Jason from the Mendelsohn lab at the Columbia University Medical Center. Compared to my first two weeks at the lab, I have been given a lot more work to do. For most of my time, I have been able to section many paraffin blocks embedded with either a mouse embryo or a mouse bladder using a rotary microtome. By cutting and collecting the important tissue, microns in thickness, we are able to take an even clearer look at the development of bladder cancer. I also have been able to stain many of my slides using the H&E staining method or by the immunostaining method, applying specific antibodies. Yesterday, I witnessed my first mouse sacking. Katya brought one of the mice to the lab in a small white box. I didn't know what was inside until she told me. I was startled when she opened the box and held the small black rodent in her hand. Then she put one hand on its neck and the other on its tail and pulled, dislocating its head. I was really freaked out but tried not to show it. She laughed at my attempt to conceal my emotions. She cut open its abdomen and took out its bladder. I was amazed. I have dissected a number of animals in the classroom, but I have never witnessed the actual sacrifice of a live animal to be dissected seconds later. It was a whole new experience for me.

I can't believe its been almost a month since I first started and the time spent at the lab has been flying by. Overall, it has been a great experience working in the lab.

(Paraffin block-mouse embryo)

(One section of the block in the water bath) 

(1 1/2 weeks of cutting and staining)

(Microtome and water bath) 

Week 1-2 at the Mendelsohn Lab

Hi my name is Jason and I am working at the Mendelsohn lab for the summer. I am beginning my second week here at the lab and already feeling more comfortable. When I arrived at the Columbia Irving Cancer Research Center and stepped into the lab, almost 10 days ago, I was nervous and really early. I got there thirty minutes early thinking that was the right thing to do, but ultimately waited for the next thirty minutes worrying that I was in the wrong building or that they had forgotten about me. Carolina, one of the researchers at the lab, arrived minutes later after my minor panic attack and I was relieved. She was told by my PI, Dr. Mendelsohn, that this was my first day and showed me to the lab. I soon got to meet all six other students and researchers. They were a mix of a medical student, graduate students, and post docs and all welcomed me to the lab. The Mendelsohn lab studies urological cell biology, specifically bladder cancer. Because bladder cancer has many variations and can only be differentiated through histology (the study of tissue through examination under the microscope), the course of treatment may be inaccurate depending on what each histologist sees. This makes potential treatment options useless if the histologist makes the wrong diagnosis. The primary goal of this lab is to find certain genetics markers in the mass growing on the bladder to determine which kind of cancer it is (Transitional cell bladder cancer, non-muscle invasive bladder cancer, invasive bladder cancer, or squamous cell bladder cancer). For the first couple days, I spent my time following, watching, and reading. I eventually learned what my primary job would be at the lab; put simply, it is to paraffin section and to stain. Paraffin sectioning is the process of cutting thin films (5nm) of tissue, in my case mouse bladder tissue or a mouse embryo, embedded into a block of paraffin wax to be then put on slides for further examination. Dan, a medical student, taught me how to paraffin section and it was very difficult at first. The thin film of tissue is so delicate and sticky that it either folds on itself or just rips apart before I could even get close to putting it into the water and onto the slide. After about two days, I got the technique down. Once I would finish the paraffin sectioning, I would move these slides for staining. I have learned two types of staining so far: ABC staining and H&E staining. I just follow the protocol and time how long I put the slides into the numerous solutions; it is very straightforward. (Actually, tomorrow I am going to learn a new type of staining technique called fluorescent staining. I don't really know what it is yet, but it sounds very interesting.) After an hour of reading if I have time, I end my day hopping on the A train and then the M66 to make my way home.

--- [I'll put up photos on the next post.]