Shufan "Vikki" Yin_Dr. Babak Mehrara_Week 2

During this week, I shadowed experiments in Dr. Mehrara's lab and learned and practiced experimental techniques, including immunofluorescence, qPCR, and flow cytometry. I further enhanced my understanding of in vivo experiments.

On Monday, I shadowed and practiced immunofluorescence staining of patient samples. Both normal and lymphedema (LE) arm tissue samples from patients with LDEX > 6.5 (indication of the development LE) were selected. The samples were already fixed with 4% paraformaldehyde.  To prepare samples for staining, we followed a hydration process. First, we immersed the samples in xylene to remove paraffin for 20 minutes, then gradually reduced the ethanol concentration and finally changed to distilled water. Meanwhile, we diluted the primary antibodies to target concentration. Following hydration, we performed antigen retrieving by immersing samples in 1x citric acid for 30 minutes at 90°C and then 30 minutes at RT. Instead of using 3% BSA, we chose goat serum for blocking. Subsequently, we used CD4, insulin receptor, and CD45 antibodies to stain for T cell, insulin resistance, and memory T cells. 

On Tuesday, I attended a lecture on OpenAI. The lecture taught us usage of AI on interpreting MRI report for disease diagnosis. In lab, I learned and practiced the procedures of qPCR for quantification of DNA in 100% and 50% confluent HUVEC and LEC. Major procedures including mRNA extraction and quantification, cDNA mix, and PCR.  However, during the experiment, we noticed that the mRNA concentration in 50% confluent HUVEC were four times higher than 100% HUVEC. This unexpected result could be due to human error during sample transfer. 

On Wednesday, I shadowed another flow cytometry experiment on cells from mice lymph nodes, ear skin, and back skin. I practiced determining the location of mice lymph node. After harvesting lymph nodes, ear skin, and back skin. The tissues were finely cut into tiny pieces for digestion and removal of red blood cells. After staining for dead cells and lymphatic endothelial cells (LECs) using DAPI, CD31 antibody, and podoplanin antibody, we used the flow cytometry machine to determine the population of LECs in lymph nodes, ear skin, and back skin. 



On Thursday, I learned tissue sectioning. The lab meeting scheduled on Friday was canceled. 


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