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Author Topic: staining caused by normal serum plus Envision  (Read 3356 times)

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Offline weneng

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staining caused by normal serum plus Envision
« on: August 25, 2005, 06:56:33 PM »
Dear all,

I have another question need ask your opinon. I expected a negative result from my normal rabbit serum control but it turned out to be dark brown everywhere. My procedure is 1% H2O2 after deparaffinzation, then DAKO serum free protein block, then normal rabbit serum. then DAKO Envision+ to rabbit. Did I do it wrong way? This is my first time doing this control. Could anybody tell me why I get this?

Thanks,
Wen

staining caused by normal serum plus Envision
« on: August 25, 2005, 06:56:33 PM »

Offline ImmunoNYC

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staining caused by normal serum plus Envision
« Reply #1 on: August 26, 2005, 12:05:05 AM »
Why did you use serum as a negative control? Did you dilute it? Is your primary a unpurified antiserum that forces you to use this as a control? I use highly purified rabbit IgG with the EnVision system.

Offline madeleine huey

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staining caused by normal serum plus Envision
« Reply #2 on: August 31, 2005, 02:08:38 PM »
Hello,
Sometime it's depending on your tissue.  My huntch is your End. Biotin is not quite well block.  Try this;  don't use the quench solution from the kit, but instead prepare 3% H202/Methanol for 30min - 60min (no harm could be done if your tissue if paraffin).  Do only 0.3% H202/MeOH for 60 min if frozen section).  Use your DAB+ chromagen for shorter time (ie. time your DAB reaction under the microscope & stop the reaction with PBS).  You can try use the same concentration rabbit isotype as your primary ab (ie. if your 1st ab is 1 ug/ml for your experiment, then you could 1ug/ml rabbit IgG also.  Rather use 5% or 10% come with the kit).
Madeleine

Offline ImmunoNYC

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staining caused by normal serum plus Envision
« Reply #3 on: September 01, 2005, 09:36:15 AM »
I use the EnVision+ kit all the time .... IMO, you definitely should be using the peroxidase from the kit, I find it to be superior to other peroxidase blockers out there.

The kit is very sensitive to too much "primary". I can rarely use above 3 ug/ml of rabbit polyclonals without background so I suspect the serum is causing the problems.

staining caused by normal serum plus Envision
« Reply #3 on: September 01, 2005, 09:36:15 AM »