Mouse kidney and small intestine were fixed with 4% paraformaldehyde, and were subsequently immersed in 30% (w/v) sucrose. Samples were blocked in PBS containing 5.0% (v/v) BSA after slides were made with a cryostat, and were subsequently incubated with our anti-KSP antibody, anti-AQP1 antibody (Santa Cruz), anti-Megalin antibody kindly provided by Dr. Sekine (Tokyo Metropolitan Institute of Medical Science, Japan) [17] (link), anti-E-cadherin antibody (Abcam) and anti-AQP2 antibody (Sigma). Alexa Fluor 488 and 594 (Invitrogen) were used as a second antibody. Nuclei were stained with DAPI (Invitrogen). Pictures were taken with TCS-SP5 (Leica) and Olympus IX81.
Mouse ES cells were stained at day 18 of the differentiation with Activin (10 ng/mL) after fixation with 4% paraformaldehyde. The samples were blocked in PBS containing 5.0% BSA and were incubated with our anti-KSP antibody conjugated with biotin and anti-E-cadherin antibody (Cell Signaling). Streptoavidin-Cy5 (Beckman Coulter) and Alexa Fluor 594 (Invitrogen) were used for the detection of KSP and E-cadherin respectively.
KSP-positive and KSP-negative cells were stained on the day after the flow cytometry. The samples were treated with Endogenous Avidin/Biotin Blocking System (Abcam) and were subsequently blocked in PBS containing 5.0% BSA and 0.1% Triton. The samples were incubated with our anti-KSP antibody conjugated with biotin, anti-human specific mitochondria antibody conjugated with biotin (Abcam), anti-Megalin antibody, anti-AQP2 (Alomone Labs) or anti-Podocalyxin (R&D) antibodies. Streptavidin-Phycoerythrin (Beckman Coulter) or Alexa Fluor 594 was used for detection.
Mouse ES cells were stained at day 18 of the differentiation with Activin (10 ng/mL) after fixation with 4% paraformaldehyde. The samples were blocked in PBS containing 5.0% BSA and were incubated with our anti-KSP antibody conjugated with biotin and anti-E-cadherin antibody (Cell Signaling). Streptoavidin-Cy5 (Beckman Coulter) and Alexa Fluor 594 (Invitrogen) were used for the detection of KSP and E-cadherin respectively.
KSP-positive and KSP-negative cells were stained on the day after the flow cytometry. The samples were treated with Endogenous Avidin/Biotin Blocking System (Abcam) and were subsequently blocked in PBS containing 5.0% BSA and 0.1% Triton. The samples were incubated with our anti-KSP antibody conjugated with biotin, anti-human specific mitochondria antibody conjugated with biotin (Abcam), anti-Megalin antibody, anti-AQP2 (Alomone Labs) or anti-Podocalyxin (R&D) antibodies. Streptavidin-Phycoerythrin (Beckman Coulter) or Alexa Fluor 594 was used for detection.
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