Immunofluorescence histochemistry is a powerful tool frequently used in neuroscience for qualitative and quantitative morphological and anatomical studies. Because samples are subjected to prolonged examination under conventional fluorescence microscopes and multiple laser-scanning confocal microscopes, it is critical for fluorescent tags to have a low susceptibility to photobleaching. Conjugates comprised of bright fluorescent tags and highly specific secondary antibodies that don't cross-react with irrelevant cell and tissue targets are optimal. Our line of NorthernLights fluorescent secondary reagents include anti-mouse, anti-goat, anti-rabbit, anti-sheep, and anti-rat affinity purified antibodies and streptavidin conjugates. These fluorochromes are bright, resistant to photobleaching, and stable in tissue clearing agents, including alcohol and xylenes. Spectral characteristics of NorthernLights conjugates are comparable to other common fluorochromes allowing use with both conventional fluorescence and laser confocal microscopes (Table 1).
| NorthernLights Secondary Reagents | Cat. # | Abs/Em Maxima | Laser (Ex) | Fluorochromes with Comparable Spectrums (Abs/Em) |
| NL-493 anti-Sheep IgG | NL012 | 493/514 |
Argon (488) | FITC (492/520) Cy™2 (489/506) Alexa Fluor® 488 (494/519) |
| NL-493 anti-Rabbit IgG | NL006 | |||
| NL-493 anti-Mouse IgG | NL009 | |||
| NL-493 anti-Rat IgG | NL015 | |||
| NL-493 anti-Goat IgG | NL003 | |||
| NL-493 Streptavidin | NL997 | |||
| NL-493 anti-Chicken IgY | NL018 | |||
| NL-557 anti-Sheep IgG | NL010 | 557/574 |
Krypton (568) HeNe (543) |
Phycoerythrin (565/575) Rhodamine Red™ X (570/590) Cy™3 (548/562) |
| NL-557 anti-Rabbit IgG | NL004 | |||
| NL-557 anti-Mouse IgG | NL007 | |||
| NL-557 anti-Rat IgG | NL013 | |||
| NL-557 anti-Goat IgG | NL001 | |||
| NL-557 Streptavidin | NL999 | |||
| NL-557 anti-Chicken IgY | NL016 | |||
| NL-637 anti-Sheep IgG | NL011 | 637/658 |
HeNe (633) | Allophycocyanin (645-660) Alexa Fluor® 647 (650-668) Cy™5 (650/670) |
| NL-637 anti-Rabbit IgG | NL005 | |||
| NL-637 anti-Mouse IgG | NL008 | |||
| NL-637 anti-Rat IgG | NL014 | |||
| NL-637 anti-Goat IgG | NL002 | |||
| NL-637 Streptavidin | NL998 | |||
| NL-637 anti-Chicken IgY | NL017 |
High fluorescence output and resistance to photobleaching make NorthernLights conjugates the reagents of choice for the detection of low abundant cell and tissue antigens. NorthernLights streptavidin conjugates can also be used for the neuronal tracing of biocytin-filled neurons. Labeled neurons in tissue slices can be viewed with exceptional resolution that details fine morphological structures in axons, dendrites, and dendritic spines. This allows for accurate 3D neuronal reconstruction and image analysis (Figure 1).
| Neuronal Tracing | |
 View Larger Image |
Figure 1. Neuronal tracing of medium spiny neurons in mouse nucleus accumbens using NorthernLights-557 Streptavidin conjugate. Low-magnification image (left) represents a Z-stack projection of 82 images collected in 1 micron steps. High-magnification image of the dendrite with spines (right) represents a Z-stack projection of 48 images collected in 0.1 micron steps. |
Acknowledgement : We express our thanks to Dr. Mark J. Thomas and researchers Said Kourrich and Patrick E. Rothwell (Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA) for providing brain slices with biocytin-filled neurons.
Cy is a trademark of GE Healthcare. Rhodamine Red is a trademark of Invitrogen. Alexa Fluor is a registered trademark of Invitrogen.