Positive immunostaining in negative control - (Sep/12/2006 )
Hi. I've been working with Vectastain Elite ABC kit for a month. First, I didn't get any signal in a positive human sample. This sample must to show some signal but my negative is very clean (beautiful) with 4 minutes of staining. So I decided test the kit with a positive control. I'm using rat (a know sample) as my positive and the same sample as negative control without primary antibody. I suppose see signal only in my positive control but I got signal in both. I can see diffuse brown in my negative and my positive. I'm using DAB to stain. I start with 6 minutes and jump to 4 minutes but I can't see any difference.
Anyone can help me, please.
Anyone can help me, please.
What concentration are you using with the ABC kit? We use a 1:100 dilution as opposed to the drop method described in the instructions. Also, it might help if you posted the protocol you are using.
we use 1:1000 dilution from the ABC kit.
The diffuse staining in both pictures is background. There is also background on your slide which leads me to believe that either your blocking buffer/step is not working effectively (what is in the buffer and how long do you block?) or your washing technique is not as stringent as it should be.
Also, when it comes to rat and mouse tissue you will tend to get more background. There are kits out there that are especially made for use on rodent tissue to decrease background. What species primary and secondary are you using? Also, your primary antibody concentration is probably too high.