COS7 cells were transfected with 6µg pcDNA-TIRC7-myc and pcDNA3-myc as control. After transfection the expression of recombinant TIRC7 protein was analyzed in transfected COS7 with pcDNA3-TIRC7-myc (COS7t) and untransfected (COS 7nt) with anti-TIRC7 mAb17. Immunostaining with anti-myc antibody (myc-Ab) (Oncogene) was used as positive control for the transfection. Cells were incubated overnight at 4°C with anti-TIRC7 mAb (10µg/ml) and anti-myc mAb´s (Oncogene,1/10) rinsed with PBS, and incubated with Cy2-conjugated goat anti-mouse IgG (Dianova,1/250) or goat anti-human IgG (Sigma, 1/100) diluted in 5% milk/PBS. Cells were mounted with 50% (v/v) glycerol/PBS and analyzed by confocal laser scanning microscopy.
Wild types and TIRC7 knockout mice splenocytes were fixed for 20 min at 4°C with 4% paraformaldehyde in PBS and permeabilized. Cells were incubated overnight at 4°C with anti-TIRC7 antibody (10µg/ml) or with anti-human IgG (hIgG, 5µg/ml, Sigma) as negative control. Cells were rinsed with PBS, and incubated with Cy3-conjugated goat anti-human IgG (1:250, Sigma) diluted in 5% milk/PBS. Cells were mounted with 50% (vol/vol) glycerol in PBS and confocal images were obtained by using Zeiss LSM 510 confocal laser scanning microscope.
Wild types and TIRC7 knockout mice splenocytes were fixed for 20 min at 4°C with 4% paraformaldehyde in PBS and permeabilized. Cells were incubated overnight at 4°C with anti-TIRC7 antibody (10µg/ml) or with anti-human IgG (hIgG, 5µg/ml, Sigma) as negative control. Cells were rinsed with PBS, and incubated with Cy3-conjugated goat anti-human IgG (1:250, Sigma) diluted in 5% milk/PBS. Cells were mounted with 50% (vol/vol) glycerol in PBS and confocal images were obtained by using Zeiss LSM 510 confocal laser scanning microscope.