Goat anti mouse igg fitc conjugated secondary antibody

Cell morphology was analyzed under an inverted phase-contrast microscope (Axiovert 200; Carl Zeiss, Oberkochen, Germany) and the images were obtained by digital camera (AxioCam HRC; Carl Zeiss). For immunofluorescence staining, cells were washed and fixed in 4% phosphate-buffered paraformaldehyde (25 minutes at 20 °C) and permeabilized with 1% Triton X-100 in PBS (15 minutes at 4 °C). After washing with PBS, the cells were treated with 5% BSA in PBS for 1 hour before incubation with primary antibodies specific for ZO-1 (Invitrogen, Carlsbad, CA, USA), α-SMA (Abcam, Cambridge, MA, USA) or β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 5% BSA overnight at 4 °C. The cells were then washed with 0.2% Tween 20 in PBS before incubation with goat anti-mouse IgG-FITC-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature in the dark. The nucleus was counterstained with DAPI (4′,6-diamidino-2-phenylindole), and the cells were visualized under the Axiovert 200 fluorescence microscope with 10X10 and 20X10 NA objectives equipped with AxioCam HRC digital camera. Digital photographs were obtained with Axiovision 4.3 (Carl Zeiss) and merged images were obtained using Photoshop 7.0 (Adobe Systems, Toronto, Ontario, Canada).

Cells were fixed with 4% formaldehyde after 0, 7, 14, and 21 days of differentiation. The fixed cells were washed, permeabilized, blocked, and incubated with albumin (1:100; Santacruz Biotechnology, Dallas, TX, USA) antibodies overnight at 4 °C. A goat anti-mouse IgG FITC-conjugated secondary antibody (1:100; Santacruz Biotechnology) was used to detect the signals. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) mounting solution (Abcam). The fluorescence signals were detected with a fluoroscopic microscope (AxioObserver Z1; Carl Zeiss, Oberkochen, Germany).

The immunofluorescent staining for high-mobility group box 1 (HMGB1), for nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) (all at 1:200 dilutions. Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were performed. The slides were incubated with each primary antibody independently overnight at 4 °C. Sections then were washed three times with PBS and incubated with goat anti-mouse IgG (FITC)-conjugated secondary antibody (1:200; Santa Cruz, Biotechnology, Inc.) at room temperature for 1 h. Afterward, sections were washed and mounted in medium containing DAPI.
The histological image captures (200× magnification) were performed using an Axio Imager.A2 microscope with an integrated camera (Axiocam ICc5, Carl Zeiss Microscopy GmbH, Jena, Germany). The percentage for TUNEL, nuclear Nrf2 positive cells, GFAP, and staining intensity for HO-1 were measured using the Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The analysis was performed in 5–8 sections from each specimen (for a total of 25 to 40 observations per group), proximal to the optical disk due to the higher density of ganglion cells. GCL, INL, and ONL were counted by two masked observers to evaluate staining interpretations. A third observer was required in cases of disagreement.