We are trying to do IHC in fresh-frozen thin sections of rat heart. Our primary antibody is raised in goat (and there are no other alternatives available). We've been using Abcam's secodary anti-goat fluorophore conjugates (Alexa 488, 594, 555) and we have almost more staining in the no-primary controls than in the sections with primary. We've tried all the standard troubleshooting techniques (different blocking conditions, temps, incubation times, etc) and still have trouble. We've talked with Abcam and they sent pre-adsorbed antibodies and we still have background!
Can anyone recommend a secondary anti-goat that has worked in rat heart sections?
Thanks in advance.
We often are using Abcam's secodary anti-goat.
We haven't anyone problem.
Excuse me.
BA-6000
Vector Laboratories, Inc.
Biotin–Rabbit anti-sheep IGg ANTIBODY
1,5 mg
€ 200
Vector Laboratories, Inc.,
30 Ingold Road, Burlingame, CA 94010 U.S.A.
Tel: (650) 697-3600 • Fax: (650) 697-0339 •
Email: [email protected] •
Web site: www.vectorlabs.com
I would prefer to use paraffin sections (Buchwalow and Boecker, 2010).
See also: (Buchwalow et al., 2011).
Buchwalow I, Samoilova V, Boecker W, Tiemann M. 2011. Non-specific binding of antibodies in immunohistochemistry: fallacies and facts. Sci Rep 1:28.
Buchwalow IB, Boecker W. 2010. Immunohistochemistry: Basics and Methods, 1 ed. Heidelberg, Dordrecht, London, New York: Springer.
We used goat antirabbit secondaries from Sigma CF488 and CF555, and from Invitrogen AF594 for rat brain paraffin sections. Results were fine. Good luck with your work.
Check out Jackson ImmunoResearch: . https://www.jacksonimmuno.com/?gclid=CKmbzKK4-swCFVhZhgodkXAKwQ
This is the go-to place for a wide variety of secondary antibodies. I have used their donkey anti goat secondaries with great success. Use the Alexa-Fluor conjugated versions for great anti-fade properties. You will also need their normal donkey serum for blocking.
Dear Lori, you can to try using anti-goat immunoglobulin ImmPRES Reagent Peroxidae, Vector. I have tried it and the staining results are satisfactory.
Good luck
Hi Lori,
I've had good luck with Sigma, Jackson and Life Technologies brand secondaries, I always buy affinity purified and cross adsorbed versions, usually with Alexa fluors. you ALWAYS need to troubleshoot the concentration of your secondary.
Of note, Life Tech/Thermo has a new type of very low background "superclonal" secondary you might try here. I haven't used this yet but may try it for some difficult tissues as it isn't much more expensive:
https://www.thermofisher.com/ca/en/home/life-science/antibodies/secondary-antibodies/superclonal-secondary-antibodies.html
Before you buy anything though, you should consider that In frozen sections, high background most often is the result of poor quality freezing technique, not antibody problems. What is your freezing method? Have you run an H&E stain to be sure that you are properly preserving you proteins and don't have cellular leakage and freezing artifact leading to this background? A good frozen section H&E should have crisp morphology and cellular detail with strong cytoplasmic Eosin staining, indicating lots of protein and morphological preservation in the cells.
Thanks everyone for the suggestions. Hopefully I'll be able to work out my conditions!
I use the Goat-on-Rodent HRP-Polymer from BIOCARE MEDICAL ref GHP516H
see www.biocare.net
4040 Pike Lane Concord,CA 94520
rgds and succes Enno
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