Tissue staining problem with polyclonal antibody - (Aug/24/2005 )
I made an polyclonal antibody for a protein. Western works great. It shows
just one band, which is gone in null tissue prep. 
However, when used for tissue staining, it displays the same patterning for both wild-type and null tissues. 
( I test the same tissue for both western and staining.)
Why is that? How could I fix the problem. Any suggestion is highly appreciated.
I can help if you tell me what are the specifics of your protocol.
For starters:
- Are you using a secondary which will not cross react with the endogenous IgG in your tissue?
- Did you block appropriately?
- Also is this an affinity purified polyclonal or just antiserum from the animal?
- Did you try different dilutions of the polyclonal?
- Do you have a positive control (like another antibody you know which will work with your protocol)?
The tissue used for staining is Drosophila eye discs.
The polyclonal antiserum is from rat. I didnot do purification.
I tried different dilutions. I am thinking about trying more should try more.
I used anti-Rat IgG secondary. It works beautifully with another antibody prepared with the same protocol.
For that very protocol, there is no blocking step.
For starters:
- Are you using a secondary which will not cross react with the endogenous IgG in your tissue?
- Did you block appropriately?
- Also is this an affinity purified polyclonal or just antiserum from the animal?
- Did you try different dilutions of the polyclonal?
- Do you have a positive control (like another antibody you know which will work with your protocol)?
The tissue used for staining is Drosophila eye discs.
The polyclonal antiserum is from rat. I didnot do purification.
I tried different dilutions. I am thinking about trying more should try more.
I used anti-Rat IgG secondary. It works beautifully with another antibody prepared with the same protocol.
For that very protocol, there is no blocking step.
The polyclonal antiserum is from rat. I didnot do purification.
I tried different dilutions. I am thinking about trying more should try more.
I used anti-Rat IgG secondary. It works beautifully with another antibody prepared with the same protocol.
For that very protocol, there is no blocking step.
Well I have never stained drosophila so I am not sure how well normal IHC methods translate over but I routinely stain mammal tissue of various species so here is my advice ... your background is likely b/c it is antiserum and not affinity or otherwise purified antibody. In that case I would recommend using a preimmune antiserum as a negative control and blocking well (a combination of BSA and serum from the animal in which your secondary was made). Definitely try more dilutions of your antiserum and block well!
Good luck!!