Technical Note: Using NorthernLights Fluorescent Secondary Antibodies for Neuronal Tracing

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
Click to view Graph
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
Click to view Graph
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
Click to View Graph
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
Neuronal tracing of medium spiny neurons in mouse nucleus accumbens using NorthernLights-557 Strept­avidin conjugate.
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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.