Immunofluroescent staining fixed tissues

February 27, 2015

Pharmacology and experimental neuroscience
University of Nebraska Medical Center
Graduate student

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Company:

Jackson Immuno Reseach

Product Name:

AffiniPure Donkey Anti-Mouse IgG (H+L) Alexa Fluor 594

Catalog Number:

715-585-150

In mouse models of Parkinson's Disease we use multiple primary antibodies to look at neuron populations following addition of MPTP. This secondary antibody has been used with anti-TH, anti-Map2, and anti-Gad67 in different sets of tissues depending on what we were looking at for that particular experiment.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Fixed brain tissue

Primary Incubation

1:500 - 1:2000 depending on primary, 48 hrs., 4 degrees

Blocking Agent

5% Normal donkey serum

Secondary Incubation

1:1000 or 1:2000 depending on primary used, 1 hour, room temp

Tertiary Incubation

NA

Detection

TexasRed fluorecent light

Results Summary

All the antibodies we have used have stained well with this secondary antibody. The signal is always bring at a higher magnification (40x), however at lower magnifications (4x) it can be difficult to distinguish antibody-fluorescence from background fluorescence. This is mainly true with Map2 and Gad67 antibodies, don't see this problem with TH primary antibodies.

Additional Notes

None

Image Gallery

Summary

The Good

Bright at high magnifications

The Bad

Can be difficult to see cell of interest at lower magnification

The Bottom Line

Works well enough to keep using. Definitely test this secondary antibody before using it to determine if it will work for your experiments.

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