Fitc conjugated secondary antibody

Caco-2 cell monolayers grown on 0.33 cm² Transwell supports were fixed with 4% paraformaldehyde in PBS for 30 min, washed, and permeabilized in 0.3% Triton X-100 in PBS. Then, they were incubated with a monoclonal antibody against Zonula occludens-1 (ZO-1; Invitrogen, Carlsbad, CA, USA), washed, and incubated with FITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) and DAPI. Stained monolayers were examined using a Leica TSC SP5 (Leica Microsystems, Wetzlar, Germany) confocal microscope equipped with × 60 objectives. Stacks were imported into AutoVisualize 9 (AutoQuant Imaging) for presentation of 3D projections and maximum volume projections were created with the 5D viewer deconvolution software.

A549, H226, and H1975 cells were trypsinized, plated into 24-well plates with slides on the bottom for 24 h culture, and washed using cold PBS. 4% paraformaldehyde was used for cell fixation for 30 min, and cells were permeabilized using 0.5% Triton X-100 for 15 min, then blocked using 3% BSA for 60 min at 20 °C. After blocking, a Rab27A mouse primary antibody was diluted to 1:200 with 3% BSA, then added to the plate and incubated overnight at 4 °C. After discarding the Rab27A primary antibody, the cells were washed using cold PBS and incubated with FITC-conjugated secondary antibody (Invitrogen) for 1 h at 20 °C. Cell nuclei were stained with Hoechst (Beyotime), and images were captured using a confocal microscope (Nikon Eclipse Ti, Tokyo, Japan).

Seven days after surgery, seven-micron-thick tissue samples were harvested from the similar position of the flaps the flap, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E). For immunohistochemistry, samples from flap tissues were snap-frozen in liquid nitrogen; seven-micron-thick frozen sections were fixed in cold acetone and stained with rat monoclonal anti-CD31 (1∶50, ab7388, Abcam, Cambridge, MA, USA) primary antibody, followed by addition of FITC-conjugated secondary antibody (1∶1000, A-11006, Invitrogen Inc., Carlsbad, CA, USA). On one hand, neovascularization was assessed by measuring the number of capillaries in 20 fields in each mouse on H&E stained slides with high power field (HP) in each group, on the another hand, vascular density was examined under fluorescence microscope (Olympus, Tokyo, Japan) and was assessed by measuring the number of CD31+ cells in frozen sections. Capillary density was assessed morphometrically by examining three fields per section of the flap in six successive sections on both the H&E sections and the immunofluorescence staining sections after immunofluorescence staining for endothelial cells with an anti-CD31 antibody. All measurements were performed by two blinded reviewers.

Cells were rinsed two times with PBS followed by 4% paraformaldehyde fixation for 30 min and permeabilizing with 0.2% Triton X-100 for 20 min. After being blocked by 5% BSA to reduce non-specific binding, the cells were incubated with primary antibodies overnight. After washing (0.1% Tween-20 in PBS), cells were incubated with FITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h. DAPI (4,6-diamidino-2-phenylindole) (Invitrogen, Carlsbad, CA, USA) was used to show the nuclei. Slides were mounted and examined under Nikon A1 confocal laser microscope system (Tokyo, Japan).

Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.3% Triton X-100 for 30 min at room temperature (RT). Non-specific binding was blocked with 3% bovine serum albumin (BSA) in PBS for 2 h at RT. Then, cells were incubated with the primary antibodies against α-SMA (dilution 1:300; Abcam) and Col1A1 (dilution 1:500; Abcam), overnight. Afterwards, cells were washed with PBS, followed by incubation with a FITC-conjugated secondary antibody (dilution 1:200; Invitrogen) for 2 h at RT in the dark. The cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA). The slides were analyzed using a laser scanning confocal microscope (Nikon, Nagoya, Japan).

hRPE cells were grown on Falcon 4-well chamber slides (Corning Inc.) and treated as described above. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes. Cells were then blocked and permeabilized with 5% goat serum (Invitrogen, Carlsbad, CA) and 0.3% Triton X-100 for 1 hour. Primary antibodies reactive against activated caspase 3 and caspase 4 (Abcam, Cambridge, MA) were added for overnight prior to addition of FITC-conjugated secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour. Slides were mounted with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Slides were examined using a confocal microscope (LSM510, Carl Zeiss, Thornwood, NY).

Sections were deparaffinized, and the antigen retrieval was performed for 20 min at 98 °C in citrate buffer (10 mM, pH = 6.0). Sections were then blocked in 2.5% Normal Goat Serum Blocking Solution (Vector) for 1 h at room temperature, followed by incubation with primary antibodies (α-SMA, 1:500; Col-1, 1:400; Fibronectin, 1:200; CD31, 1:200; PCNA, 1:200; Arg1, 1:200; CD206, 1:900; iNOS, 1:50; TGFβ1, 1:100; F4/80, 1:200; LIGHT, 1:200) overnight at 4℃. For IHC, the biotinylated secondary antibody (Vector) was applied, and the signal detection was performed using diaminobenzidine (DAB) solution (Vector). For immunofluorescence, sections were incubated with FITC-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).