Table 1.
Multiple fluorescence labeling.
| First primary antibodies | First visualizing immunoreagents | Second primary antibodies | Second viualizing immunoreagents |
|---|---|---|---|
| Immunolabeling combined with the Cy2-staining of WFA-binding sites in mice, rats and sheep* | |||
| rabbit-anti-collagen IV (1:100; Merck Millipore, Billerica, MD, USA) | Cy3-donkey-anti-rabbit IgG | guinea pig-anti-Iba (1:100; Synaptic Systems, Göttingen, Germany) | Cy5-donkey-anti-guinea pg IgG |
| rabbit-anti-collagen IV (1:100; Merck Millipore) | Cy3-donkey-anti-rabbit IgG | guinea pig-anti-GFAP (1:200; Synaptic Systems) | Cy5-donkey-anti-guinea pig IgG |
| guinea pig-anti-parvalbumin (1:200; Synaptic Systems) | Cy3-donkey-anti-guinea pig IgG | rabbit-anti-collagen IV (1:70; Merck Millipore) | Cy5-donkey-anti-rabbit IgG |
| guinea pig-anti-parvalbumin (1:200; Synaptic Systems) | Cy3-donkey-anti-guinea pig IgG | rabbit-anti-aggrecan (1:100; Merck Millipore) | Cy5-donkey-anti-rabbit IgG |
| Immunolabeling combined with the Cy2-staining of WFA-binding sites in rodents* | |||
| sheep-anti-neurocan (1:200; R&D Systems, Wiesbaden-Nordenstedt, Germany) | Cy3-donkey-anti-goat IgG | rabbit-anti-aggrecan (1:200; Merck Millipore) | Cy5-donkey-anti-rabbit IgG |
| goat-anti-versican (1:20; R&D Systems) | Cy3-donkey-anti-goat IgG | rabbit-anti-aggrecan (1:100; Merck Millipore) | Cy5-donkey-anti-rabbit IgG |
| goat-anti-hHAPLN1 (1:100; R&D Systems) | Cy3-donkey-anti-goat IgG | rabbit-anti-aggrecan (1:100; Merck Millipore) | Cy5-donkey-anti-rabbit IgG |
| rabbit-anti-Kv3.1b (1:1000; Härtig et al., 1999) | Cy3-donkey-anti-rabbit IgG | guinea pig-anti-parvalbumin (1:300; Synaptic Systems) | Cy5-donkey-anti-rabbit IgG |
| rabbit-anti-Kv3.1b (1:1000) | Cy3-donkey-anti-rabbit IgG | guinea pig-anti-NeuN (1:200; Synaptic Systems) | Cy5-donkey-anti-guinea pig IgG |
| rabbit-anti-parvalbumin (1:500; Synaptic Systems) | Cy3-donkey-anti-rabbit IgG | guinea pig-anti-NeuN (1:200; Synaptic Systems) | Cy5-donkey-anti-guinea pig IgG |
*In general, biotinylated WFA (15 µg/ml; Vector) and mixtures of primary antibodies were concomitantly applied overnight followed by cocktails of Cy2-streptavidin, Cy3- and Cy5-tagged secondary antibodies. All fluorescent immunoreagents were obtained from Dianova (Hamburg, Germany) and used at 20 µg/ml for 1 h. Abbreviations: WFA, Wisteria floribunda agglutinin; Iba, ionized calcium binding adapter molecule 1; GFAP, glial fibrillary acidic protein; hHAPLN1, human hyaluronan and proteoglycan link protein (cartilage link protein 1, CRTL); NeuN, neuronal nuclei.