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.