Most of the Antibody company (having pr. Ab conc. range 0.2 mg/ml - 0.5mg/ml) websites suggests dilution around 1:200, but it seems not staining or faintly staining. What is the hand on experience on bench for scientists performing IHC/ICC?
Dear Dr. Chandra Kishore
The correct dilution of primary antibody for IHC/ICC is best determined by first selecting a fixed incubation time and then making a series of dilutions in a titration experiment.
For instance, for IHC, if the data sheet suggests dilution of 1:200, I would not go by it because the experimental conditions are different. I would be interested in that dilution that works best for my tissue/cells. So, I will have to find the optimal concentration of the primary antibody for IHC/ICC to determine the optimal signal with low background noise.
But from the dilution recommended by the company I would know (more or less) which dilutions I should work on for IHC/ICC. So, I would try dilutions of 1:50, 1:100, 1:200, 1:400, and 1:500. Each dilution should be performed on the same type of sample in order to retain the same experimental conditions.
Please note that most of the antibodies will have comparable batch-to-batch consistency, in which case only one titration experiment would be required. However, especially for polyclonal antibodies, there could be a possibility that the results of staining may change between batches. In such a case, another titration experiment would be necessary.
Regards,
Malcolm Nobre
In addition to Malcom's advice, you may also need to experiment with several other parameters including antigen retrieval method and chromogen development time. It's not always a simple process, but companies usually provide their own tests so you have an idea of what you might expect using those conditions.
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