I used an antibody against H2-Kb SIINFEKL tagged with APC on Dendritic cells treated with nanocarriers carrying SIINFEKL peptide. After 3 hour incubation, the cells were washed 3 times with PBS and fixed for 20mins in 10% neutral buffered formalin. The cells were wash again with PBS, and blocked with 1% BSA in PBS for 1 hour at room temp. The antibodies were then added to the cells diluted in the above blocking buffer, overnight at 4 degrees. The cell were then washed 3 times in 1X PBS and counterstained with Hoechst and AF488- WGA (Nucleus and cell membrane respectively). The problem is I expected to visualize the MHC class I loaded with SIINFEKL, on the cell membrane. Instead much of the staining is on the lysosome (i did another experiment and confirmed that the peptides are indeed in lysosomes and the staining pattern looks similar), and the signals are quite strong. But i did not permeabilize the cells. The control untreated cells with no nanocarriers look clean, with no background stain at all. How did the antibody enter the cell without permeabilization?
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering
- Coating