Double or triple immunofluorescence staining was used to analyze multiple markers within the same section. Free-floating sections were washed three times for 10 min each in Tris-Triton buffer, pH 7.4 before being incubated overnight at 4°C under continuous agitation with the primary antibodies raised in different species (Table 1) in a solution containing 2% Triton X-100 and 2% NGS in Tris-Triton buffer, pH 7.4. The next day, the sections were again rinsed three times for 10 min with Tris-Triton buffer, pH 7.4 followed by incubation with secondary antibodies raised in goat against the different species of the primary antibodies and coupled to either Alexa Fluor 488, Cy3, or Alexa Fluor 647 [or the plant lectin Wisteria floribunda agglutinin (WFA) coupled to Cy3 for staining of PNNs] in a solution containing 0.5 μl DAPI and 2% NGS in Tris-Triton buffer, pH 7.4 at RT for 30 min in the dark. After another washing step of three times 10 min with Tris-Triton buffer, pH 7.4, sections were mounted on gelatin-coated glass slides and cover-slipped with Dako fluorescence mounting medium (Dako).

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