IHC problem with a polyclonal AB - (Dec/17/2005 )

Hi ....

I ran a goat polyclonal Wnt7a antibody on wild-type and null mice .... they both showed positive staining ... my -ve controls came clear of any staining .... What could I possibly do to avoid this problem?

Please any help/suggestions would be highly appreciated


Thanks

O believe the question might be if polyclonal antibodies are appropriate in this case, in westerns we can reduce unspecific binding by augmenting the concentration of BSA in the bloking solution but with your IHC maybe you can tell me if there is any similar approach.
Is the intensity of te signal the same in the negative control?

Maybe you can reduce the dilution of your primary antibody to a minimum

Hi ....

I ran a goat polyclonal Wnt7a antibody on wild-type and null mice .... they both showed positive staining ... my -ve controls came clear of any staining .... What could I possibly do to avoid this problem?

Please any help/suggestions would be highly appreciated


Thanks

Hi!

Well, I´m with 'tertu'. Might be a problem to use a polyclonal in IHC in this case. As I don´t know your protocols: what kind of controls other than wild-type and null-mice do you have?
I mean Santa Cruz antibodies can really suck sometimes and can show a lot of unspecific signals in western-blot without recognizing the actual protein.

What kind of secondary antibody do you use, have you tried different dilutions of your first and second antibody? Sometimes it is well worth to do a 'secondary antibody control' alone just to see what kind of staining you get. Are the staining patterns and signal intensities similar in your wt and null samples?

Cheers

Hi tertu & Bomber.... Thanks for your inputs ...

main reason to use polyclonal was: Not much Wnt Ab's are out there, and for Wnt7a Santa Cruz is the only one avalible through vendors to date.

the tissues were teeth and uterus from each the Wt and the null, in addition a -ve ctrl without 1ry Ab was used on Wt teeth .... didn't use an internal -ve ctrl (thought the null tissue would serve the purpose) .... I used a vectastain anti-goat 2ry ... blocking was with a rabbit serum (where the secondary was obtained)

Results: no stain on the -ve ctrl ( excluded binding of 2ry to non-specific signals)
both wt and null tissue showed a close patteren and intensity of stain (weaker on the null though). it proved that the reaction is mainly due to the 1ry Ab

Next step would be to tailor down the dilution of 1ry Ab,although I did that previously on Wt tissue alone to get optimum dilution.
I have the feeling that the problem is that the 1ry Ab is cross-binding another molecule. was hoping to explore the problem to take precautions, besides I might be missing an integral step I'm not aware of.... hopefully you guys would no how to go around this problem.

Best regards

Maybe you should do a western blot of your wt tissue (as well as null), just to make sure the polyclonal binds only to the expected protein.
Of course that's a problem with polyclonal: you may bind to more epitopes, thus more likely to have spurious binding to unrelated antigens.
Is the antibody used at optimal concentration, btw?
Good luck.

ZEZO ... If you give more details of your protocol I will be glad to help your troubleshoot. Can you give me the basic run-down of the steps of staining. Questions I have:

Frozen or paraffin?
What block do you use?
What concentration primary?
How long primary incubation?
What secondary? Concentration? Incubation time?
What mode of detection??

Hi MaximinaNYC ....

1. Formalin fixed decalcified paraffin embeded

2. I used a rabbit serum block from vectorstain kit for 30min at room temp

3. I tailored my Ab to a 1:250 (its a goat anti-mouse polyclonal)

4. I incubate my primary overnight at 4C

5. secondary is an anti-goat made by vector, 50ul of 2ry + 150ul blocking + 10ml PBS , 200ul for 30min

6. detected using DAB

Any Advice would be highly appreciated.

N.b:
* I preadsorbed my Ab with 10% mouse serum for 2 hrs at 37C - slightly better result but still unspecific staining
* I used a blocking peptide from the vendor as a -ve ctrl and no staining

Hi Zezo, I am not a fan of Vector kits at all so unless you are willing to buy new reagents I may not be able to help, but I will try.

- What concentration is your primary ... not what dilution???
- Is this kit a biotin kit? Do you block biotin if so?
- Have you blocked biotin?
- Might include BSA in your blocking solution.
- I think because the blocking peptide did as it should and blocked the staining it means that likely the non-specific staining is due to the antibody which makes me think perhaps the concentration of primary is simply too high. Have you titrated it on a positive control prep??

Why not consider another company's anti-goat? I use antibodies from Jackson ImmunoResearch with great success. I can help you with a protocol.
Also, maybe your protocol is perfect and you are running into the same problem A LOT of people have with these antibodies from Santa Cruz. They cross react with other proteins and when doing western it is easy to elucidate b/c of size but on IHC it is extremely difficult and it appears as "background". You may want to call the company if you haven't already.

Hi MaximinaNYC .... Thanks for giving my problem a lot of your attention ... really appreciate it.

- the concentration is 0.2 ug/ul ... I did titrate this previously to end up with a dilution of 1:250... samples where I diluted to 1:500 showed no reaction.

- Yes the kit is Avidin/Biotin ... and didn't block against biotin knowing that our tissue doesn't contain any ( I might be wrong here) ... but -ve ctrls always comes dead clear.

- didn't include BSA up to this point as blocking... but I did block with a combination of rabbit serum (where the 2ry was raised) + mouse serum (the host tissue I'm running the 1ry on) ... What conc. of BSA would you recomend?

- budget is a problem here ... but since you recommend Jackson's anti-goat ... let me see around probably someone wouldn't mind to spare littile ... but from your experience what makes Vector not a very good one ?

- your last point is what I certainly fear ... I heard the news on Santa Cruz ... They claim its specific .. and no other epitope shares a close homology ... they immediatly sent me a new lot (give them credit on the speed of tech. support) results were the same if not worst should of done a Westeren to confirm the specificity of Ab but I haven't ... Simply because I don't have the tools ... I'm working on resources

awaiting for your feedback ... I'm glad to share experience and get your advise, if it doesn't work at the end (am starting to loose hope) at least will carry all the good points for the future.

Kind regards,
Zezo