Table 1.
Double and triple fluorescence labeling.
| First marker Primary antibodies |
First marker Secondary antibodies∗ |
Second marker Primary antibodies/lectin |
Second marker Secondary reagents∗ |
Third marker Primary antibodies/lectin |
Third marker Secondary reagents∗ |
|---|---|---|---|---|---|
| Rabbit-anti-claudin 11/OSP (1:400; 241 003, Synaptic Systems, Göttingen, Germany) | Cy3-donkey-anti-rabbit IgG (711-165-152) | Guinea pig-anti-MBP (1:200; 295 004; Synaptic Systems) | Cy2-donkey-anti-guinea pig IgG (706-225-148) | ||
| Rabbit-anti-NG2 (1:100; AB5320; Merck Millipore, Billerica, MA, United States) | Cy2-donkey-anti-rabbit IgG (711-225-152) | Guinea pig-anti-MBP (1:400; 295 004; Synaptic Systems) | Cy3-donkey-anti-guinea pig IgG (706-165-148) | ||
| Rabbit-anti-serum albumin (1:200; 286 003; Synaptic Systems) | Cy2-donkey-anti-rabbit IgG (711-225-152) | Goat-anti-collagen IV (1:100; AB769; Merck Millipore) | Cy3-donkey-anti-goat IgG (705-165-147) | Biotinylated STL (20 μg/ml; B-1165; Vector, Burlingame, CA, United States) | Cy5-streptavidin (016-170-084) |
| Rabbit-anti-MBP (1:400; 295 002; Synaptic Systems) | Cy3-goat-anti-rabbit IgG (111-165-144) | Biotinylated STL (20 μg/ml; Vector) | Cy2-streptavidin (016-220-084) | ||
| Rabbit-anti-claudin 11/OSP (1:400; 241 003; Synaptic Systems) | Cy3-donkey-anti-rabbit IgG (711-165-152) | Guinea pig-anti-MBP (1:200; 295 004; Synaptic Systems) | Cy2-donkey-anti-guinea pig IgG (706-225-148) | Mouse-anti-CNP 1 (clone 335C6, 1:200; 355 011; Synaptic Systems) | Cy5-donkey-anti-mouse IgG (715-175-151) |
∗All fluorescent immunoreagents were obtained from Dianova (Hamburg, Germany) as supplier for Jackson ImmunoResearch (West Grove, PA, United States) and used at 20 μg/ml for 1 h. OSP, oligodendrocyte-specific protein; MBP, myelin basic protein; NG2, neural/glial antigen 2 proteoglycan; STL, Solanum tuberosum lectin (= potato lectin); CNP 1, 2′,3′-cyclic nucleotide-3′-phosphodiesterase.