I am iontophoretically injecting BDA (biotinylated dextran amine) into the motor cortex for anterograde axonal tracing. My BDA is tagged with Alexa594. I'm doing 5 minute injections with a current of 5 uA, alternating 7 seconds on and 7 seconds off with pulled glass pipettes of 20-50 um. I'm having really nice staining of the axons but the BDA also stains the vessels near my injection site. Is there any way to avoid this or is this just an inevitable issue with the method?
I've attached a picture. Has anyone encountered this before?
vessel staining usually is because of endogenous peroxidases. Pre-incubation with H2O2 will eliminate usually 10ml of 3% solution brought up to 50 ml.
Non-specific staining of blood vessels may be due to improper or insufficient perfusion of the brain.
Vessels are effectively very enriched in endogenous peroxidase, so you have to perform a treatment before beginning IHC procedures (use H2O2 as suggested by Malinda, 20 min pretreatment or a mixture of methanol-H2O2).
Inactivation of endogenous peroxidase with H2O2 does not improve non-specific labelling when you are doing immunofluorescence. Endogenous peroxidase reacts with hydrogen peroxide to reduce the 3,3'-diaminobenzidine (DAB) substrate or other peroxidase substrates, resulting in non-specific staining of the tissue.
You're right Afsaneh. I've not paid attention to the details of staining in the question. However, I don't think that the quality of fixation is determinant concerning the high back ground staining observed in vessels
I don't see any lingering blood in my vessels when I'm sectioning and my brains are appropriately bleached so I'm unsure if it could be a perfusion problem. Thanks for the suggestion though!
Dear Katherine, did you find the answer? I get similar artefacts (not always) with dyes but lately also with viruses. It is striking as many publications using such approach do not show such artefacts. I did high magnification scans and these are individual cells within the blood vessels. I am wondering if that depends on the pulse, current or the affinity of the dye or virus towards these cells
Anna Stepien - I never did find a good solution with BDA. I had better luck with viruses (AAV, PRV) as tracers and have been doing pressure injections instead of iontophoretically injecting tracers/viruses. Best of luck with your labeling!
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