Chicken embryo fibroblasts (CEFs) were prepared from 9 day old embryonated eggs at The Pirbright Institute as described previously48 . CEF, DF-1 (immortalized chicken fibroblasts) and MDCK cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin and streptomycin (P/S) at 37 °C in 5% CO2 incubator. The Drosophila Schneider 2 (S2) cells were maintained in the Schneider’s Insect Medium (Sigma-Aldrich, Dorset, UK). AMV-3C2-S (gag) antibodies were purchased from Hybridoma Bank of Iowa, University of Iowa. Antibodies against nucleoprotein (NP) of influenza virus and the fusion (F) protein of NDV were raised in mouse as described previously49 (link), 50 (link). Alexa-flour 568 secondary antibodies were purchased from Invitrogen Carlsbad, CA, USA and IRDye 800CW α-mouse secondary antibody were acquired from LI-COR, Nebraska USA.
Alexa flour 568 secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-flour 568 secondary antibodies are fluorescent-labeled antibodies used in immunodetection techniques. They are designed to bind to primary antibodies, allowing for the visualization and localization of target proteins or antigens in biological samples.
Automatically generated - may contain errors
3 protocols using alexa flour 568 secondary antibodies
1
Preparing Chicken Embryo Fibroblasts for Experiments
2
Preparation and Characterization of Chicken Embryo Fibroblasts
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CEFs were prepared from 9-day-old embryonated eggs at The Pirbright Institute as described previously42 . Immortalized chicken fibroblasts (DF-1), human embryonic kidney cells 293T (HEK-293T) and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin and streptomycin (P/S) at 37 °C in 5% CO2 incubator. AMV-3C2-S (gag) antibodies were purchased from Hybridoma Bank of Iowa, University of Iowa. α-V5 and J2 antibodies for the detection of V5 tag and dsRNA were from Genetex, and SCICONS, respectively. Alexa-flour 568 secondary antibodies were purchased from Invitrogen Carlsbad, CA, USA and IRDye 800CW α-mouse secondary antibodies were acquired from LI-COR, Nebraska USA. Poly I:C (a synthetic analogoue of dsRNA), dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS), were purchased from Invivogen and Sigma whereas chicken IFN-β was produced in HEK-293T cells43 .
3
Embryonic Tissue Immunohistochemistry Protocol
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Embryos harvested for immunohistochemistry (IHC) analysis were fixed with 4 % PFA for 3 hours at room temperature. Embryos were then embedded in Shandon M1 embedding matrix media (Thermo Scientific) and quickly frozen over dry ice. Mounted embryos were sectioned on Leica CM1850 cryostat and consecutive 20 µm thick sections were collected on positive-charged glass slides (Globe scientific). Antibody staining was performed following standard protocols on slides stacked in Shandon Sequenza slide rack (Thermo Scientific) and supported by Shandon cover plates.
Primary antibodies against anti-mouse PAX6, PAX7 and NKX6.1 were obtained from development Studies Hybridoma Bank. Anti-chicken NGN2 antibody was a kind gift from Dr.
Bennett Novitch (Skaggs et al., 2011) . Rabbit polyclonal antibody against GFP Tag was obtained from AnaSpec Inc. Goat anti-mouse Alexa Flour 488, Alexa Flour 556 and goat anti-guinea pig Alexa Flour 568 secondary antibodies (Invitrogen) were used for detecting primary antibodies.
Sections were covered with DAPI-containing mounting media (Vectashield) and a cover slip, and sealed with nail polish.
Primary antibodies against anti-mouse PAX6, PAX7 and NKX6.1 were obtained from development Studies Hybridoma Bank. Anti-chicken NGN2 antibody was a kind gift from Dr.
Bennett Novitch (Skaggs et al., 2011) . Rabbit polyclonal antibody against GFP Tag was obtained from AnaSpec Inc. Goat anti-mouse Alexa Flour 488, Alexa Flour 556 and goat anti-guinea pig Alexa Flour 568 secondary antibodies (Invitrogen) were used for detecting primary antibodies.
Sections were covered with DAPI-containing mounting media (Vectashield) and a cover slip, and sealed with nail polish.
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