Alexa fluor 647 conjugated f ab 2 goat anti mouse igg h l secondary antibody

Transfected HEK293T cells on chambered glass slides were prepared for immunofluorescence as previously reported (20 (link), 35 (link)). Briefly, fixed and permeabilized cells were blocked for 60 minutes in blocking solution (1% BSA in 1× PBS) and then incubated at 4°C overnight with either rabbit anti-PDE6C (71 (link)) or mouse anti-HA antibodies diluted in the blocking solution. After washes with PBS, cells were incubated in the dark for 1 hour in either Alexa Fluor 568–conjugated goat anti-rabbit (Life Technologies, A-11011) or Alexa Fluor 488–conjugated goat anti-mouse (Life Technologies, A-11017), or Alexa Fluor 647–conjugated F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific, A-21237) diluted in the blocking solution. After washes with PBS, where indicated, the nuclei were counterstained with To-Pro-3 (Thermo Fisher Scientific) for 30 minutes in the dark at 25°C. Cells were mounted using Vectashield mounting medium (Vector Laboratories, Inc.) and imaged using Plan-Neofluar ×40/1.3 oil lens and a LSM 510 confocal microscope (Zeiss).

To examine whether feline PD-1 binds to PD-L1/PD-L2, 2 × 10⁵ fePD-1–EGFP- and fePD-L1–EGFP-expressing cells were incubated for 30 min with 10 μg/mL of either fePD-1–Ig, fePD-L1–Ig, or fePD-L2–Ig at room temperature (RT), followed by another 30 min incubation with Alexa Fluor 647-conjugated F(ab′)₂-goat anti-Rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific). Rabbit IgG (Southern Biotech) was used as a control protein. Cell fluorescence was analyzed using BD FACSLyric system (BD Biosciences, Franklin Lakes, NJ, USA).
To examine PD-L1 expression on cell lines, 2 × 10⁵ cells were incubated for 30 min with 10 μg/mL CL1Mab-7 or mouse IgG₁κ isotype-matched control antibody (15H6; Southern Biotech) at RT, followed by another 30 min incubation with Alexa Fluor 647-conjugated F(ab′)₂-goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific). Cell fluorescence was analyzed using BD FACSLyric system (BD Biosciences). For fePD-1–EGFP- and fePD-L1–EGFP-expressing cells, only EGFP-positive cells were gated and subjected to further analysis.