I am currently working on stem cell markers (5 markers) on cervical cancer patient tissue. Some one suggested me to run all the markers together for one tissue then proceed with next tissue sample.
OR
I can run one whole batch of samples with one antibody then other.
Will it make any difference?
Dear Nidul, I normally prefer to perform the staining for a marker on a whole batch of samples and then to proceed with the next marker, always on the whole batch of samples. I think is better to perform the same immunostaining on all the samples within the same experiment in order to be able to evaluate the different protein expression and localization between the samples being able to rule out that these are related to different experimental procedures. Generally, in the same experiment we charge all samples to be tested, 1 negative and 1 positive control for each marker. If the sample number is too high, you can perform immunostaining for one marker on an appropriate number of samples at a time and, in the second experimental set perform the same staining on the other samples but including a slide of the first set. This will help you to be sure that the staining procedure you follow gives the same result. I hope this is helpful. Let me know
I would also recommend to stain one antibody after the other. From technical point of view it is better to know first, that the marker works on all types of tissue. If one of it fails because of the need of a different AB-titer (eg high expression vs low expression), you have to restain all your specimens with the new titer for a valid comparison.
And there are usually less variations, if you provide one larger amount of working solution instead of serveral times making a small amount.
Do you work on FFPET? Have in mind, that the prepared sections for IHC should be stained within maximal 2 weeks. Too long storage can influence your antigens.
Dear Nidul
Theoretically it does not make any difference whether to run separate or together. However, I would prefer to process each antibody on the different samples in a single run together with the positive and negative controls to eliminate the possibility of confounding environmental factors and inter-process variations (temperature, incubation time, amount of wash etc). This would also allow you to examine your results and get an preliminary reflection on whether there is a difference in the expression between the different groups. Otherwise, you will have to wait to all groups are processed and ending with a load of analysis at the same time.
Regards,
Bassem
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