Rhodamine conjugated donkey anti rabbit igg

HeLa or U2OS cells were fixed with ice-cold methanol for 30 minutes at −20°C, and immunofluorescence microscopy was performed as described previously (O'Regan and Fry, 2009 (link)). Primary antibodies were against rootletin (1∶200, this study; or 1∶100, Santa Cruz, sc-67824), C-Nap1 (1∶1000, Fry et al., 1998a (link)), Nek2 (1∶200, BD Transduction Labs, 610594), GFP (0.5 µg/ml, Abcam, 6556), Cep135 (1∶1000, Kim et al., 2008 (link)), Myc (1∶1000, Cell Signaling Technology, 2276), γ-tubulin (1∶1000, Sigma T6557), cyclin B1 (0.2 µg/ml, Abcam, 2949) and pC-Nap1 AQDL and LLEK (0.5 µg/ml, this study). Secondary antibodies used were Alexa-Fluor-488-conjugated and Alexa-Fluor-594-conjugated goat anti-mouse-IgG and goat anti-rabbit-IgG (10 µg/ml, Invitrogen A11001 and A11012, respectively) or Alexa-Fluor-488-conjugated donkey anti-goat-IgG and Rhodamine-conjugated donkey anti-rabbit-IgG (5 µg/ml, Jackson 705-545-003 and 711-025-152, respectively). Images were captured using Volocity software (Improvision) on an inverted Nikon TE300 or Olympus BX51 microscope with a 100× oil objective. Intensity measurements were quantified using Volocity or ImageJ (v1.4.1) software and images were processed in Adobe Photoshop 4.0.

MSC-induced CECs were washed three times with PBS, fixed in 10% neutral buffered formalin overnight in a refrigerator (2–8 °C), and then blocked with 0.1% bovine serum albumin (Sigma-Aldrich) and 5% donkey serum (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) at room temperature for 30 min. MSC-induced CECs were then incubated with primary antibodies for zonular occludens 1 (ZO-1; 1:100, 61-7300; Life technologies), alpha 1 sodium-potassium ATPase (ATP1A1; 1:500, ab76020; Abcam, Inc., Cambridge, MA, USA), CD166 (1:200, 559260; BD Biosciences, San Jose, CA, USA), NCAM-1/CD56 (1:200, MAB24081; R and D systems, Minneapolis, MN, USA), and N-cadherin (1:150, ab5581; Abcam, Inc.) overnight at 4 °C. Cells were washed three times with PBS and incubated with rhodamine-conjugated donkey anti-rabbit IgG and/or FITC-conjugated donkey anti-mouse IgG (1:100; Jackson ImmunoResearch Laboratories Inc.). Cells were again washed three times with PBS for 10 min each and stained with 4’-6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, CA, USA) for 1 min to counterstain cell nuclei. The fluorescence signals were detected with either a laser-scanning confocal microscope (Olympus, Tokyo, Japan) or a fluorescence microscope (EVOS, Life Technologies).