Alexa fluor 568 goat anti rabbit igg h l

NMuMG cells were grown on coverslips, fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 for 15 minutes at room temperature. Subsequently, cells were blocked for 20 minutes at room temperature in blocking solution (10% goat serum, 5% FCS and 0.5% BSA in PBS) followed by incubation with primary antibodies at 4°C overnight. These samples were washed three times with PBS for 10 minutes at room temperature following incubation with fluorochrome-labeled secondary antibody or Phalloidin-633 as well as Hoechst for DNA counterstain (20ng/ml) for 1 hour at room temperature. For mounting of the samples Immu-Mount (Thermo Scientific) reagent was used and samples were imaged with a confocal laser-scanning microscope (SP05, Leica). Data were processed with ImageJ software.
Antibodies used for immunofluorescence were E-Cadherin (13-1900, Invitrogen and 610182, BDTransduction Laboratories), N-Cadherin (610921, BD Transduction Laboratories), ZO-1 (617300, Invitrogen), Alexa Fluor-488 goat anti mouse IgG (H+L) (A11029, Invitrogen); Alexa Fluor-568 goat anti rabbit IgG (H+L) A11011 Invitrogen; and Alexa Fluor-633 goat anti rat IgG (H+L) A21094, Invitrogen, Alexa Fluor 633 Phalloidin (A22284, Invitrogen) was used to stain F-actin.

Cells were grown on plastic chamber slides, fixed in 4% Paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min and blocked for 15 min in 10% of Fetal Bovine Serum (FBS) in PBS. Cells were incubated with α-tubulin (Invitrogen, 1:1000) and NGFR (Advance Targeting systems, 1:100) antibodies in 1% FBS in PBS and incubated 2 h at room temperature (RT). Secondary antibody Alexa Fluor 568 goat anti-rabbit IgG (H + L) (Invitrogen) was used at 1:1000 in PBS (10% FBS) and incubated for 45 min at RT. Glass cover slips were mounted with VECTASHIELD mounting medium with DAPI H-1200 (Vector Laboratories). Images were taken with the confocal microscope Zeiss AxioObserver Z1.