실험 Q&A Immunology > Immunofluorescence
세포 면역 염색에서 두가지 이상의 일차 항체를 사용할 때
레벨1 biobibibi (대학원생)
안녕하세요 immunofluorescent stain을 처음 진행하게 되는데 관련 학과 학생도 아니라 어려움을 느껴 도움을 받고자 글을 올리게 되었습니다.
1. 세포 고정 후에 immunofluorescent staining을 진행해야 하는 것으로 알고있는데, reference에서는 동일한 부분을 0h와 36h에 관찰하였습니다. 다른 부분을 관찰한 것인지, 특정한 protocol이 있는지 알고 싶습니다. ( cell tracking을 사용한 것인가요..?)
2. 동일한 부분 일차 항체(Marker)를 두가지 이상 사용할 때 어떤 프로토콜을 사용해야 하는지 알고싶습니다. 보통 일차 항체를 O/N 하던데, 두가지 마커를 사용하려면, O/N를 두번하고 각각에 맞는 이차 항체를 염색하면 될까요?
Immunostaining of EMT markers on A549 spheroids at 0 h and 36 h in co-culture with HUVECs. A–D. Expression of vimentin in spheroids at 0 h and 36 h. Blue: nuclei; green: vimentin. E–H. Expression of E-cadherin in spheroids at 0 h and 36 h. Blue: nuclei; green: E-cadherin.
Cell culture media was removed from the devices and samples in the microfluidic devices were first rinsed in cold PBS and then at fixed in 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) for 15 min room temperature. Then 0.1% Triton-X (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated for 5 min before blocking by Block Ace (Dainippon Phamaceutical, Osaka, Japan) for 2 h. To demonstrate the endothelial cell monolayer formation, staining of VE-cadherin (1:100, mouse; Sigma Aldrich, USA) was carried out. For analysis of epithelial marker expression, E-cadherin (1:100, mouse; Sigma Aldrich, USA) was used to stain for cell-cell junctions; and nuclei were stained with DAPI (Sigma-Aldrich, USA). Fluorescent images were obtained using FluoView 1000 confocal microscopy (Olympus, Japan). The secondary antibody used was 2mg/ml Alexa Fluor 488-conjugated anti-mouse IgG antibodies (Invitrogen, USA). For analysis of vimentin expression, vimentin (1:200, rabbit; Invitrogen, USA) was used and incubated again with 2 mg/ml Alexa Fluor 488-conjugated anti-rabbit IgG antibodies (Invitrogen, USA), and DNA was labeled by Hoechst (Invitrogen, USA). Fluorescent images were obtained using a phase-contrast microscope equipped for fluorescence (Nikon, Japan).
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