Alexa fluor 568 donkey anti rabbit igg antibody

Embryonic progenitor and adult-derived cells, exposed to differentiation medium for 14 days at confluence, were washed once with PBS and fixed in 4% paraformaldehyde for 30–60 min at room temperature (RT). Fixed cells were washed three times with PBS, permeabilized and blocked by incubation in blocking buffer (5% normal donkey serum, 1% BSA and 0.1% Triton X-100 in PBS) for 1 h at RT. The cells were then incubated overnight at 4 °C with primary rabbit anti-human UCP1 polyclonal antibody (Thermo Sci. PA1-24894) at a dilution of 1:500 in 5% normal donkey serum, 0.5% BSA and 0.05% Triton X-100 in PBS. Then, the cells were washed four times with PBS plus 0.05% Triton X-100 (PBS-Triton) and incubated for 1 h at RT with Alexa Fluor 568 donkey anti-rabbit IgG antibody (Invitrogen, A10042) at a 1:500 dilution in PBS-Triton. Isotype controls were stained under identical conditions except that total rabbit IgG (Life Technologies, 10500C) was used as primary antibody (at the same concentration with UCP1 Ab, 1.34 μg/ml). Cells were counterstained with DAPI at 0.1 ng/mL for 10 min at RT and imaged on a Nikon Eclipse TE2000-U inverted microscope.

For detection of CXCR4 and CD31, cells were washed once with PBS and fixed in 4% paraformaldehyde for 30–60 min at room temperature (RT). Fixed cells were washed three times with PBS, permeabilized, and blocked by incubation in blocking buffer (5% normal donkey serum, 1% BSA, and 0.1% Triton X-100 in PBS) for 1 h at RT. The cells were then incubated overnight at 4 °C with CD31 monoclonal antibody with Alexa Fluor 488 (Thermo Sci. MA5-18135, Waltham, MA, USA) and primary rabbit anti-human CXCR4 polyclonal antibody (Thermo Sci. PA1-24894, Waltham, MA, USA) at a dilution of 1:500 in 5% normal donkey serum, 0.5% BSA, and 0.05% Triton X-100 in PBS. Then, the cells were washed four times with PBS plus 0.05% Triton X-100 (PBS-Triton) and incubated for 1 h at RT with Alexa Fluor 568 donkey anti-rabbit IgG antibody (Invitrogen, A10042, Waltham, MA, USA) at a 1:500 dilution in PBS-Triton. Isotype controls were stained under identical conditions except that total rabbit IgG (Life Technologies, 10500C, Carlsbad, CA, USA) was used as primary antibody. Cells were counterstained with DAPI at 0.1 ng/mL for 10 min at RT and imaged on a Nikon Eclipse TE2000-U inverted microscope.

Plasmids encoding FLAG-tagged wild-type and Q99L mutant of RagB, the lentiviral vector pWPI, the lentiviral envelope plasmids pMD2.G and psPAX2 were obtained from Addgene. The plasmids encoding wild-type and S16H mutant of Rheb were gifts of Dr. Bo Xiao (Johns Hopkins University School of Medicine) and were described previously [25] . Leucine, UA, the FLAG M2 antibody, and Cocktail 3 were from Sigma Aldrich. The Anti-LAMP1 antibody was from ABCAM. Alexa Fluor 488 Donkey anti-Mouse IgG (H+L) antibody and Alexa Fluor 568 Donkey Anti-Rabbit IgG antibody were from Invitrogen. Protein A-Sepharose beads were from GE Healthcare Life Sciences. Clarity Western ECL substrate was from bio-rad. HRP labeled anti-mouse and anti-rabbit secondary antibodies were from Promega. Normal rabbit IgG and normal mouse IgG were from Millipore; the RagB antibody was from Proteintech. All other antibodies were from Cell Signaling Technology.