Goat anti mouse fitc conjugate igg

Cells seeded on collagen-coated coverslips (1×10⁵ cells per mL) were washed 3 times with PBS for 5 minutes and fixed in 4% paraformaldehyde for 10 minutes. The fixed cells were then washed with PBS for 3×5 min and incubated in a blocking buffer (10% goat serum [Invitrogen, USA] 0.3% Triton X-100 [Fluka, USA]) at room temperature for 30 minutes. They were then incubated at 4°C overnight with the following primary antibody mouse anti-nestin monoclonal antibody (1:200, Millipore, USA). The next day, the cells were rinsed for 3×5 min to remove unbound primary antibodies. Subsequently, they were incubated at room temperature for 2 hours with the following conjugate secondary antibody: goat anti-mouse FITC-conjugate IgG (1:1400, Abcam, UK). The cell nuclei were counterstained with 1 μg/mL 4, 6-diamidino-2-phenylindole (Sigma-Aldrich, USA) in PBS in the dark at room temperature for 1 min. After washing, the samples were mounted on a slide with mounting media for visualization using a fluorescence microscope. To examine the specificity of the nestin antibody, 3T3 fibroblast-like cells (Pasteur Institute of Iran, Tehran) were used as a negative control. The labeled cells were identified using fluorescent microscopy (Olympus Ax70).

About 48 to 72 hours before cell transplantation, BrdU (5 μmol/mL; Sigma-Aldrich, St. Louis, MO, USA) was added to the flask of cultured cells. For checking cell labeling with BrdU, 48 h after cell labeling, the labeled cells on collagen-coated coverslips were washed in PBS for 3×5 min and fixed in 4% PFA for 10 min. Then, the fixed cells were washed in PBS for 3×5 min and incubated in 2N HCl at 60°C for 45 min, and were washed 2 times in 0.1 M borate buffer (pH 8.3). After being washed in blocking buffer (10% goat serum, Sigma-Aldrich, USA/0.3% Triton X-100 Fluka, USA and 1% BSA) at room temperature for 60 min, the incubated cells were again incubated with the primary antibody anti-BrdU (1:500, Sigma-Aldrich, USA) at 4°C overnight. The next day, the cells were rinsed in PBS for 3×5 min to remove unbound primary antibodies. Subsequently, they were incubated at room temperature for 1 h with the secondary antibody of goat anti-mouse FITC conjugate IgG (1:200, Abcam, Cambridge, UK), washed in PBS for 3×10 min, mounted with mounting media, and visualized using a fluorescence microscope (Figure 1C).

Hair follicle stem cells isolated from the bulge region were incubated with every 100 mL primary antibody, mouse anti-CD34 monoclonal antibody (1:75, Sigma, USA), and mouse anti-nestin monoclonal antibody (1:200, Millipore, USA) at room temperature for 1 h. The cells were centrifuged with 1–2 mL of Phosphate-Buffered Saline (PBS) (0.1 M).
Subsequently, the cells were incubated in the dark at room temperature for 1 h following conjugate secondary antibody: goat anti-mouse FITC-conjugate IgG (1:1400, Abcam, UK). After this time, the percentage of CD34+, K15, and nestin+were analyzed by flow cytometry.