Alexa fluor 488 donkey anti chicken igy igg h l

Monkey embryos at day 7 were fixed in 4% paraformaldehyde and 0.1% polyvinyl pyrrolidone (PVP) in PBS (pH 7.2–7.4) for 20 min at room temperature. They were then washed three times in PBS and incubated with 0.3% Triton X-100 and 0.1% PVP for 30 min at room temperature. After washing three times in PBS, embryos were blocked for 2 h in blocking buffer (3% BSA + 10% FBS + 0.1% PVP) and separately incubated overnight at 4 °C with the following primary antibodies: chicken anti-GFP (Abcam, ab13970, 1:500), mouse anti-Oct4 (Santa Cruz, SC5279, C-10, 1:400), and goat anti-Gata6 (R&D Systems, AF1700, polyclonal, 1:400). Embryos were washed three times in PBS/0.05% Tween-20 and incubated with the following secondary antibodies for 2 h at room temperature Alexa Fluor 488 Donkey Anti-Chicken IgY† (IgG) (H + L) (Jackson Immunoresearch, 703-545-155, 1:500), Alexa Fluor 568 donkey anti-mouse IgG Thermo Fisher Scientific, Waltham, MA, USA A-10037, 1:600), and Alexa Fluor 647 donkey anti-goat IgG (Thermo Fisher Scientific, Waltham, MA, USA, A-21447, 1:600). DAPI (Roche Life Science, Basel, Switzerland, 10236276001, 1:1000) was used to stain nuclei.

Human synovium, cartilage or mouse knee joint samples were fixed in 4% paraformaldehyde overnight at 4 °C. Mouse knee joints were decalcified in 10% EDTA (pH 7.4) for 5–7 days. All samples were dehydrated in 30% sucrose for 1 day at room temperature and sectioned at 10 μm using the CryoJane tape-transfer system (Leica). Sections were incubated in blocking buffer (PBS with 5% horse serum) for 1 h at room temperature and then stained with anti-FAP (R&D, AF3715, 1:500), anti-OLN/CLEC11A (R&D, BAF1904, 1:500), anti-Oln/Clec11a (R&D systems, AF3729, 1:500), anti-beta galactosidase (GeneTex, GTX77365, 1:500) or anti-Col2a1 (Boster, BA0533, 1:200) antibodies in blocking buffer overnight at 4 °C. Slides were washed 3 times with PBS and then stained with Alexa Fluor 647 donkey anti-Sheep IgG (H+L) (Thermo Fisher Scientific, A21448, 1:500), Alexa Fluor Plus 555 donkey anti-goat IgG (H+L) (Thermo Fisher Scientific, A32816, 1:500) or Alexa Fluor 488 donkey anti-chicken IgY (IgG) (H+L) (Jackson ImmunoResearch, 703-545-155, 1:500) secondary antibodies in blocking buffer for 1 h at room temperature. Slides were then washed 3 times with PBS, stained with 1 μg/ml DAPI for 1 min and mounted with Anti-fade Prolong Gold (Invitrogen). Images were acquired using an Olympus IX73 microscope.

After the indicated time post infection, the cells on glass slides were fixed with paraformaldehyde 4% for 10 min, washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 5 min (for H7 extracellular staining cells were not permeabilized) and incubated with antibodies against NP (catalogue number: PA5-32242; Thermo Fisher; 1:2000), H7 (ref. 44 (link)) (1:100), or M1 (provided by Dr. Jovan Pavlovic; 1:10) for 1 h at room temperature. After washing with PBS and incubation with corresponding secondary antibody Cy3 goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch; diluted 1:200), Alexa Fluor 488 donkey anti-chicken IgY (IgG) (H+L) (Jackson ImmunoResearch; diluted 1:500) or Cy3 goat anti-mouse IgG (H+L) (Jackson ImmunoResearch; diluted 1:200) for 30 min at room temperature, nuclei were stained for 5 min with DAPI (diluted 1:10,000 in PBS). The glass slides were examined on an Axioplan 2 Imaging System (Carl Zeiss, Oberkochen, Germany) equipped with an ApoTome.