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Author Topic: antigen blocking for HIF-1 /Dako LSAB link  (Read 6955 times)

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Offline lostUCLA

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antigen blocking for HIF-1 /Dako LSAB link
« on: February 17, 2007, 01:47:08 AM »
   Hi, everyone.
   
  I'm staining cells from nasal lavage fluid with anti HIF-1 alpha (rabbit polyclonal) and using the Dako LSAB+ visualization kit.  The secondary antibody (Link) in this kit is diluted in PBS. 
  Do I need antigen blocking?
  I've been having background staining.  I'm using 2% BSA in PBS to block, and I'm diluting my primary antibody (HIF-1) in BSA as well, because that's the way they told me to do it, but I have a feeling this is not correct. 
   What do you think? 

antigen blocking for HIF-1 /Dako LSAB link
« on: February 17, 2007, 01:47:08 AM »

Offline ImmunoNYC

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Re: antigen blocking for HIF-1 /Dako LSAB link
« Reply #1 on: February 19, 2007, 01:58:18 PM »
Not sure what you mean by "antigen blocking". Could your background be due to endogenous biotin? Recently we used this kit to stain and got bad background. Then we inserted biotin blocking and all background went away.

Offline lostUCLA

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Re: antigen blocking for HIF-1 /Dako LSAB link
« Reply #2 on: February 21, 2007, 04:28:57 PM »
 
 
  Antigen blocking, we do that with albumin from normal swine, bovine or goat serum.  It's supposed to block antigens in general before your primary antibody, so that you don't get nonspecific staining.  But I read in the Dako manual that you're supposed to use serum from the same species as the link or secondary antibody, but never from the species your primary antibody comes from.
   I guess you're right.  Endogenous biotin or avidin could be the reason.  What I did is I used ovalbumin as antigen blocking, and I diluted my primary antibody in 2% BSA, but I'm still getting nonspecific staining. 
   You think I could use ovalbumin and skimmed powder milk to block biotin and avidin?
    Thanks.

Offline lostUCLA

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Re: antigen blocking for HIF-1 /Dako LSAB link
« Reply #3 on: February 22, 2007, 02:58:29 PM »

  Today I'm trying Dr. Rodney Miller's recipe with egg-whites and skim milk. 
   I hope it works.

Re: antigen blocking for HIF-1 /Dako LSAB link
« Reply #3 on: February 22, 2007, 02:58:29 PM »