Abstract:
Immunohistochemistry (IHC) staining of GPR17, CHGA, GIP and GLP1 in human gut tissue and HEK-293 cells transfected with HA-tagged human GPR17 gene. Images were acquired and analyzed using LSM-700 confocal microscope and its software ZEN (ZEISS, Oberkochen, Germany) and Image J (NIH). All images are CZI formats and require ZEN software (ZEISS) to open.
Human gut samples were obtained from two cadavers at IU Health Methodist Hospital. They are male and aged at 26 and 30 years old. To determine the cell types of GPR17 expressing cell in human gut, we performed immunohistochemistry (IHC) staining by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (general endocrine cell marker; SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (marker for K cells; ab30679; abcam, Cambridge, MA) and GLP-1 (marker for L cells; NBP1-50697; Novus). Section slides were imaged and analyzed using LSM-700 confocal microscope and its software ZEN (ZEISS, Oberkochen, Germany).
To validate the specificity of GPR17 antibody, we stained GPR17 (NLS4229; Novus) and HA (#901513; Biolegend, San Diego, CA) in HEK293 cells transiently transfected by pcDNA3.1-HA-hGPR17 plasmids. HEK293 cells were transfected with pcDNA3.1 or pcDNA3.1-HA-hGPR17 plasmids for overnight. Chamber slides were imaged using LSM-700 confocal microscope (ZEISS). 10-22 randomly selected cells per image and total 10 images were quantified for staining intensity by using Image J (NIH).