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The sections were incubated with FITC-conjugated Donkey or CY3-conjugated Donkey secondary antibodies or a mixture of both for double staining.
Cy3-conjugated secondary antibody (Jackson) was used for double staining.
For double staining, antibody reactions were repeated with the second set of primary and secondary antibodies.
For double staining of GFP and BrdU, sections were first processed for the GFP immunostaining.
For double staining, directly conjugated antibodies were used in the last incubation step.
For double staining of Foxo1 and AGRP, we used an anti-FOXO1A antibody (ab12161, abcamR, Cambridge, UK), respectively.
For double staining, sections were incubated with a polyclonal rabbit antibody against synoviolin (Abgent) followed by biotinylated anti-rabbit immunoglobulins, and streptavidin-peroxidase (DAKO, Glostrup, Denmark).
This finding supports our interpretation that the structures are located directly inside nervous endings (for double staining of the dendrites with anti-neurofilament antibodies see [32]).
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The reagents used for the staining were Polymer HRP Detection System PV-90022, ZSGB-BIO) for single-staining and DouSP KIT-99999, MXB) for double-staining.
For double-staining with PNA, Alexa fluor 488-conjugate PNA was added to the secondary antibody solution.
Gated cells were then analyzed for double-staining with CD20 and IL-17.
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