Alexa fluor 488 568 conjugated donkey anti mouse

Sections were dewaxed and then subjected to heat-induced epitope retrieval with preheated epitope retrieval solution (10 mM citrate buffer, pH 6.0). The primary Abs were antibodies cocktail. Goat anti-IL-17 (R&D Systems) was used to detect IL-17 + cells, a panel of antibodies reactive with CD4, CD20, CD57, CD68, CD34 (1/2 working solution, all from Beijing Zhongshan Golden Bridge Biotech), CD4 (1:20; Leica, Germany) and MCT (1:800, Abcam, UK) were used. The sections were then incubated overnight in 4°C. In the next day, a mixture of secondary antibodies (Alexa Fluor 488/568-conjugated donkey anti-mouse or Cy3/Alexa Fluor 488 donkey anti-goat [all from Invitrogen]) were applied and incubated in 37°C for 1 hour. Nucleus were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Images were captured under a fluorescence microscope (Olympus BX51, Japan) coupled to a CCD camera (Nikon DS-Ri1) and analyzed by NIS-Elements BR 3.2 software.

For immunofluorescence, similar methods were used, except that endogenous peroxidase activity was not blocked and the primary Ab used was an antibody cocktail. Goat anti-IL-17 and Goat anti-IL-17R (both from R&D Systems, USA) were used to detected IL-17⁺ cells and IL-17R expression. A panel of antibodies reactive with CD3, CD20, CD34, CD56, c-kit (mouse monoclonal, all from Beijing Zhongshan Golden Bridge Biotech, China), CD4 (mouse monoclonal, Leica, German), CD66 (mouse monoclonal, BD Pharmingen, USA), CD68, FoxP3 and mast cell tryptase (MCT) (mouse monoclonal, all from Abcam, USA) were used. After incubation overnight at 4°C, sections underwent incubation with a mixture of secondary antibodies: Alexa Fluor 488/568-conjugated donkey anti-mouse or Cy3/Alexa Fluor 488-conjugated donkey anti-goat or Alexa Fluor 488 donkey anti-rabbit (all from Invitrogen, USA) at 37°C for 1 h. Slides were mounted with Vectashield containing DAPI (Vector Laboratories, USA) and visualized using a fluorescence imaging microscope (Olympus BX51, Japan) coupled to a CCD camera (Nikon DS-Ri1). The images were analyzed using the NIS-ELements BR 3.2 software.
Negative controls, in which PBS was used in place of the primary antibodies, were included for each marker.