Donkey anti rabbit igg alexafluor 647

T24 cells were cultured on glass cover-slips in 24-well plates, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Cells were then blocked with 10% FBS (Gibco) in PBS for 30 min at room temperature. Cover-slips were incubated with a primary antibody against Ki-67 (Maixin-Bio, Fuzhou, China). Donkey anti-rabbit IgG Alexa Fluor 647 (Molecular Probes, Waltham, MA USA) was used as the secondary antibody. Cells were further washed in PBS and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI for counterstaining. Cells were analyzed by fluorescence microscopy.

Diabetes-mimicking αTC1-6 cells were generated as mentioned above and cultured on coverslips. Experiments were done in the presence or absence of lysosomal inhibitor, BFA1, in serum-free DMEM containing 16.7 mM glucose and 0.1% BSA for 2 h. Cells were then washed 3X in PBS, fixed in 2% paraformaldehyde for 30 min, washed 5X in PBS, and incubated with blocking buffer (2% BSA in PBS containing 0.05% Tween 20) for 1 h. Cells were then incubated with appropriate primary antibodies prepared in blocking buffer against glucagon (mouse anti-glucagon antibody, Cat # ab10988, Abcam; 1:1000 or rabbit anti-glucagon antibody, Cat# ab92517, Abcam; 1:25), Stathmin-2 (goat anti-SCG10 antibody, Cat # ab115513, Abcam; 1:250), or Lamp1 (mouse anti- Lamp1 antibody, Cat # ab25630, Abcam; 1:50). Following an overnight incubation at 4°C, coverslips were washed with PBS, and incubated for 2 h in the dark at RT with appropriate secondary antibodies (donkey anti-mouse IgG Alexa Fluor 488, Cat# A-21202, Molecular Probes; donkey anti goat IgG Alexa Fluor 555, Cat# A-21432, Molecular Probes; donkey anti-rabbit IgG Alexa Fluor 647, Cat# A-31573, Molecular Probes). Then, coverslips were washed with PBS, stained with DAPI, and mounted on glass slides using ProLong antifade mountant. Image acquisition and analysis was done as described above. Experiments were repeated four times using freshly thawed cells.

Frozen tissue sections stored in −80 °C were thawed in RT to be fixated with 3% freshly made paraformaldehyde in TBS for 10 min in RT. Tissues were then permeabilized for 10 min in TBS+ 0.1%Triton-X100, rinsed three times in TBS for 5 min/ rinse. Blocking with 2% bovine serum albumin in TBS for 2 h was performed before the tissues were incubated with primary antibodies overnight at 4 °C. After rinsing with 3 × 5 min with TBS the tissues were incubated with the secondary antibodies donkey anti-mouse immunoglobulin G (IgG)-AlexaFluor 568 (1:500 Molecular Probes) and donkey anti-rabbit IgG-AlexaFluor 647 (1:500 Molecular Probes) for 1 h at RT in darkness. DNA was counterstained with DAPI (Molecular Probes) and slides were mounted with Prolong Gold (Molecular probes). Tissues were stained with antibodies against SPINK1 (1:50, H00006690-M01, 4D4, Novus), TFF3(1:200, HPA 035464, Sigma), SPON2 (1:100, A-10, st cruz), PGC (1:50, NBP1-91011, Novus), NPY (1:100, ab48789, Abcam), Aquaporin (1:100, ab168387, Abcam), NR4A1 (1:100, ab48789, Abcam), ACPP (1:100, Biologicals), P63(1:150, ab53039, Abcam), Vimentin (1:150, ab8069, Abcam). Fluorescence images were obtained with a Zeiss LSM 780 inverted confocal microscope, using a Plan Apochromat 20 × /NA (numerical aperture) 0.7 objective. Tiled images were acquired from optical sections of 5 micrometer.