Alexa fluor 488 or alexa fluor 594 labeled secondary antibodies

Briefly, four-μm-thick paraffin-embedded kidney tissue sections were deparaffined, rehydrated, and antigen repaired as described previously [9 (link)]. Next, the sections were permeabilized with 0.3% TritonX-100 for 20 min and then blocked with 5% BSA in PBS buffer for 60 min at room temperature. After incubated with primary antibodies at 4 °C overnight, the sections were washed with PBS and incubated with Alexa Fluor-488- or Alexa Fluor-594-labeled secondary antibodies (Abcam) at room temperature for 1 h. DAPI (SouthernBiotech) was used to stain nuclei.

Coverslips were coated with poly-L-lysine overnight at room temperature. Cells were rinsed with PBS, fixed in 4% paraformaldehyde for 10 minutes at room temperature, and permeabilized with PBS containing 0.25% Triton X-100. Cells were blocked in PBS-Tween (PBST) containing 10% serum from the species in which the secondary antibody was raised for 30 minutes and incubated with the indicated primary antibodies in PBST in a humidified chamber at 4°C overnight. Cells were then incubated with Alexa Fluor 488- or Alexa Fluor 594-labeled secondary antibodies (Abcam, Cambridge, MA, USA) for 1 hour at room temperature in the dark. Phalloidin-iFluor 594 (Abcam) was used to detect F-actin. After the slides were washed and sealed, the cells were imaged using inverted microscopy (TE2000-U; Nikon).