Rhodamine red x goat anti mouse

The cells were fixed in 4% PFA for 10 min and washed three times in washing buffer. After washing three times in washing buffer (PBS + 0.25% Triton X-100 + 025% BSA), the slides containing astrocytes were blocked in blocking solution (PBS + 0.25% Triton X-100) for 30 min at room temperature. Incubation with the corresponding antibodies diluted in blocking solution was done overnight at 4 °C. The following primary antibodies were used: primary polyclonal rabbit anti-MIF antibody (1:200; Abcam ab7207), primary polyclonal rabbit HTRA1 (1:200, A kind gift from Michael Ehrmann at Universität Duisburg-Essen, Essen, Germany), primary polyclonal chicken anti-GFAP (1:1.000; Abcam, ab4674), polyclonal rabbit anti-FGF8 (1:200, Abcam, ab81384) and polyclonal mouse anti-FGF18 (1:200, Abcam, ab169615). Incubation with the secondary antibodies was performed in the dark at room temperature for 1 h. The secondary antibodies were as following: Alexa Fluor-488 goat anti-rabbit IgG (1:400; Invitrogen, 828814), Rhodamine Red-X goat anti-chicken (1:400; Jackson Immuno Research, 93951) and Rhodamine Red-X goat anti-mouse (1:400; Jackson Immuno Research, 94085). The slides were washed in PBS and stained with DAPI for 20 min before mounting. For all staining procedures, negative control antibodies were performed.

After collection, pancreatic tissues were fixed in 4% phosphate-buffered formaldehyde for 24 h. Fixed tissues were then embedded in paraffin according to standard procedures [79 (link)]. Representative pancreatic slices (4 µm) were prepared and rehydrated before double immunofluorescence staining was carried out, using mouse monoclonal anti-insulin antibody (1:100,000; Sigma-Aldrich, Steinheim, Germany) and rabbit polyclonal anti-5-HT antibody (1:500; ImmunoStar, Hudson, NY, USA). Primary antibodies were detected with fluorophore-labeled secondary antibodies using Rhodamine Red-X goat anti-mouse (1:200; Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor488 goat anti-rabbit (1:400; Jackson ImmunoResearch, West Grove, PA, USA) as well as DAPI (1:1000; KPL, Gaithersburg, MD, USA). Images were recorded with an upright microscope Eclipse Ni-E (Nikon, Düsseldorf, Germany), equipped with a DS-Fi3 Color Camera (Nikon, Düsseldorf, Germany) and analysis software NIS elements AR 5 (Nikon, Düsseldorf, Germany).