Alexa flour 633 conjugated goat anti rabbit igg

For immunofluorescence microscopy, cultured cells were washed twice with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min at room temperature, and then permeabilized by incubation for 15 min with 0.1% Triton X-100 in PBS. The samples were blocked with 1% bovine serum albumin followed by incubation with rabbit anti-Smad 2/3 or rabbit anti-β-catenin (Abcam, Ltd., Cambridge, UK) primary antibody overnight at 4 °C. One day after incubation, cells were washed with PBS and incubated with Alexa Flour 488-conjugated goat anti-rabbit IgG (invitrogen, Carlsbad, CA) or Alexa Flour 633-conjugated goat anti-rabbit IgG (invitrogen) secondary antibody, respectively, for 60 min at room temperature. The final antibody treatment also contained tetramethylrhodamine isothiocyanate-conjugated phalloidin (Sigma) for actin and DAPI for nucleus staining (both at 1 µg/mL, Sigma). The slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and cells were viewed under a confocal laser-scanning microscope (LSM510, Carl Zeiss MicroImaging, Thornwood, NY).

Keloid tissue sections (n = 4) were deparaffinized, rehydrated, blocked with 5% goat serum, and incubated with mouse anti-mortalin monoclonal antibody (C1-3) or rabbit anti-p53 (sc-6243; Santa Cruz biotechnology) primary antibody overnight at 4 °C. Day after incubation, sections were washed with phosphate-buffered saline (PBS) and incubated with Alexa Flour 488-conjugated goat anti-mouse IgG (A11001; Invitrogen, Carlsbad, CA) or Alexa Flour 633-conjugated goat anti-rabbit IgG (A21070; Invitrogen) secondary antibody for 60 min at room temperature. Tissues were mounted on slides using Vectashield^® mounting medium containing the nuclear stain DAPI (Vector Laboratories, Burlingame, CA), and viewed by confocal microscopy (LSM700, Olympus, Center Valley, PA).