If I stain for the same type of tissue, but it is taken from animals at different ages, is it necessary to have age-matched isotype controls for each stained section?
I wonder if aged-matched controls would make sense, or it is just a way to waste samples, especially when I have rare samples ...
Thank you very much for sharing your knowledge and advice!
I would not run isotype controls for all the samples. One per group/age class should suffice. If there is notable non-specific staining, you should refine your assay as opposed to running more isotypes. The only thing the IgG/isotype control shows is the background induced by the secondary AB and detection system.
Hi Mohamed Mahdy , hi Andrew Pierce
Thank you very much for your kind answer/suggestion. I just want to look at some markers and see how it changes during post-natal development of mouse. In my setting, I use frozen sections of growth plate cartilage, and I have the tissue samples at 5 different ages/time points of development.
I agree with Andrew, but what I concern is that at different ages, the extracellular matrix content would also be different (the matrix would be denser, cells from mature animals would produce more matrix), so I do not know if this difference in the matrix it would affect the staining. That´s why I wonder if it makes sense to have one age-matched isotype per age group (so in total, I would have 5 isotype controls for 5 age groups).
The strategy of one isotype per age group would be a great place to start. If some ages look like they have a lot of background, then run all from that group to look at the background for each individual. Since this is developmental and not an induced immune-realted disease or similar, I would imagaine that the BG will be low.
Regarding the isotype controls, if I've understood Andrew's comment correctly, I don't think that it is true that an isotype control only shows background from the secondary. If you use an isotype control then I think that this tells you if you have non-specific binding of the primary antibody. Non-specific binding of the secondary would still show up, but I'm not sure that you can separate that from non-specific primary. Hence, a diluent only will give you the additional information to determine non-specific binding of the secondary, as it can't be from the primary.
To simplify, I think that an isotype control is A (primary) + B (secondary) = C (background), but it doesn't tell you the respective values for A and B, only that one or the other is involved.
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