Alexa fluor 488 goat anti rabbit antibody

Cells with a inoculum density of 1 × 10⁵ cells/well were cultured onto coverslips (24-well plate, Fisher Scientific) pre-coated with collagen and fixed with 4% PFA after treatments as described above. The cells were washed with PBS 2 times and incubated with 0.3% Triton X-100 in PBS (PBS/Triton) at room temperature for 10 min. For β-actin staining, the treated cells were incubated with Phalloidin, CF488A (1:100 dilution in PBS, Biotium, Inc., Fremont, CA, USA) at room temperature for 1 h. The cells were then washed three times with PBS and mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). For ZO-1 staining, cells were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h and then incubated with an anti-rabbit ZO-1 polyclonal antibody (cat. # 61-7300, 1:100 dilution, Thermal Scientific, Ottawa, Canada) at 4 °C overnight. The cells were then washed three times with PBS and incubated with an Alexa fluor 488 goat anti-rabbit antibody (Thermal Scientific, cat. # A-11034) for 1 h at room temperature. Rinsed cells were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc.). The images were taken by a Zeiss Fluorescence Microscope (Carl Zeiss Ltd., Toronto, ON, Canada) [10 (link)].

Cells were cultured onto coverslips, and they were fixed with 4% paraformaldehyde (Pfaffl) after the different treatments, following by the blocking with 5% goat serum for one h at room temperature. And then, cells were incubated with primary antibodies like the anti-rabbit ZO-1 polyclonal antibody (61-7300, 1:100, Thermal Scientific) or Phospho- NF-κB p65 (Ser536) monoclonal antibody (MA5-15160, 1:600, Thermal Scientific) at 4°C overnight. Then, after being washed with PBS three times, cells were incubated with an Alexa Fluor 488 goat anti-rabbit antibody (A32731, 1:1000, Thermal Scientific) for 1 h at room temperature. Cells were rinsed with PBS three times and then were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc.).
For actin staining, fixed cells were rinsed with PBS 3 times, and cells were permeabilized with 0.5% Triton X-100 in PBS at 4°C overnight, following by incubation with Phalloidin, CF488A (94538,1:100, Biotium, Inc., Fremont, USA) at room temperature for 1 h. Rinsed cells were then mounted with Vectashield mounting medium with DAPI, and images were took using a Zeiss fluorescence microscope (Car-Zeiss Ltd., Toronto, ON, Canada).

Eye-antennal discs from wandering third instar larvae were dissected, and fixed in 4% paraformaldehyde in Phosphate-Buffered Saline (PBS), and stained following the protocol. The primary antibodies used were mouse anti-Chaoptin (MAb24B10) (1:100, DSHB #24B10, Iowa, IA, USA), mouse anti-22C10 (1:100, DSHB #22C10, Iowa, IA, USA) and rabbit anti-GFP (1:1000, ThermoFisher Scientific #A11122, Waltham, MA, USA), while the secondary antibody were Alexa Fluor 568 goat anti-mouse antibody (1:1000, ThermoFisher Scientific #A11031, Waltham, MA, USA) or Alexa Fluor 488 goat anti-rabbit antibody (1:1000, ThermoFisher Scientific # A11008, Waltham, MA, USA).
The brains of three-to-five-day-old mtt-Gal4 > UAS-mCD8::GFP flies were dissected with their retina preserved. At first, the whole flies were fixed in 4% formaldehyde at 25 °C for 2 h. Then, the dissected brains were washed in PBT (PBS containing 0.2% Triton X-100) and blocked in 5% goat serum in PBT (PBST) for 30 min at room temperature. The primary antibodies were used to incubate the samples overnight at 4 °C. Finally, samples were washed three times with PBT and incubated overnight with Alexa Fluor 488-labeled goat anti-rabbit or Alexa Fluor 568-labeled goat anti-mouse secondary antibody overnight at 4 °C. Images of mounted samples were captured under confocal microscope (Leica TCS SP5, Wetzlar, Germany).