Donkey anti goat secondary antibody

The human melanoma tissue microarray (TMA number ME1004A) was purchased from US Biomax, Inc. (Rockville, MD, USA). The melanoma TMA was deparaffinized and antigen retrieval was performed using extended CC1 treatment (Cell Conditioning Solution, Ventana Medical Systems, Oro Valley, AZ). The goat polyclonal antibody for AXL (R&D) was applied and incubated at 37°C for 1 or 2 hours. Donkey anti-goat secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was then applied and incubated at 37°C for 60 min, followed by chromogenic detection using the DAB Map kit (Ventana Medical Systems, Oro Valley, AZ). Slides were counterstained with Hematoxylin and dehydrated and cleared before coverslipping from Xylene. RUNX2 staining was done as previously described [14 (link)].

293T cells were transfected with Invitrogen™ ViraPower™ Lentiviral Packaging Mix as followed: 2×10⁶ 293T cells were seeded the day before and mixed with 1ml Optimal MEM transfection solution with 45μl Genejuice (Millipore) containing 3.75μg pCMV-dR8.91, 2.5 μg pMD2.G-VSVG, and either 4.17 μg CHC16-pHR_hACE2_mCherry, CHC17-pHR_SARS-CoV-2 Swt_EGFP or CHC-18-pHR_SARS-CoV-2 S-Δ19_EGFP at RT for 15 mins, and then incubated with fresh D10 medium (10% FBS in DMEM without antibiotics) at 37°C and 5% (v/v) CO₂ for 12 hours. Transfected 293T cell media was changed after 24 hours and incubated for another 48–72 hours. The lentivirus supernatant was harvested, filtered (0.45 μm filter Millipore) and transduced into 293T, A549, HepG2, and SK-Hep1 cells with serum-free DMEM for 12 hours. The transduced cell media was changed with fresh complete antibiotic-containing D10 medium for another 48–72 hours. Transduced cells were flow-sorted by GFP/mCherry expression, or protein expression determined by anti-Spike protein RBD domain antibody (rabbit IgG, Sino Biological) or anti-hACE2 antibody (goat IgG, R & D Systems) followed by fluorophore-conjugated goat anti-rabbit (Invitrogen) or donkey anti-goat secondary antibody (Jackson Immuno Research). Co-culture transduced cell lines were performed as listed in Table 1.