hi,

follow the concentration of the manufacturer's recommended or from previous study. If you can not get,  you have to know is  this antibody pure or culture supernatant? then try to do titration. 

Hi,

what do you mean by dilution? Normally you have to test which concentration of the antibody fits best for your experiment. As far as i know manufacturers of antibodies give general guidelines regarding dilution. Dilutions can range from 1:50 to 1:5000 (at least which i know of).

Sincerely

Start with a good control, not your test material, and process the control tissue as you would your experimental tissue. Then titer the antibody and beware of background staining. Share the slides with a mentor. Remember that you are not guaranteed a result no matter what the product literature says - lots of bogus product out there. Look to the literature for proven reagents (that also come with a blue print for how to use them). Validate the method before experimenting or you will waste tons of time and money.

Good luck!

The manufacturer's data sheet only provides useful information as far as the clone and what was used as the immnuogen, what species it was raised in and what species it is expected to work with. The dilution factor provided is only a guide.

What I normally do is take the manufacturer's recommended dilution, multiply it by 5, take the first number you think of between 1 and 10 and divide. Then take the square root and multiply by 37.; in other words, do not pay much attention to it. We are using an antibody that has a manufacturer's recommended dilution of 1:250. We have it working successfuly, under our conditions, at 1:1600 (under a different retrieval regimen - more on this below).

Each and every antibody will have a different dilution factor as it pertains to your tissue, your preanalytical factors, your detection system and how you are performing your IHC (manual vs automated).

In addition to titering the antibody, you need to determine the best retrieval system for the antibody, again under your conditions. Just because a manufacturer recommends a particular retrieval (if in fact they do) does not mean that that retrieval is best for your conditions (e.g. we have many antibodies that work better, and with a higher dilution factor, in EDTA pH9 when the manufacturer recommends citrate, pH6.) Does the antibody even need retrieval?

I totally agree with Mark as to being careful in selection of your antibody source, and certainly with CD133  which, from experience, we have not found a reliable source - indeed many manufacturers have now retired it from their offerings. I have never had any interaction with PureGene so cannot comment on any of their antibodies.