So...I am running immunohistochemistry assays of ovarian cancer tissues that are fixated and prepared by someone else (same person every time). Lastly, between different runs with the auto stainer I have had two of the same slides (very same cut) run in two different times with the same concentration and the same program (those were the positive control) and there is a difference in staining between the two slides. This shouldn't be the case since the operator is the same, the procedure was the same and the dilution was the same. Also, same batch of antibody, so I have no clue of what can possibly be going on and how to fix it.
As paraffin dry over time, the unmasking step could be less effective after weeks even at 4°C, you can extend the unmasking time but in fact you could not compare intensities between different runs it's not relevant.
I meant what kind of staining machine do you use. For example, if you work with a Roche-ventana stainer, the prep-kits may have some issues. Or you work with a Dako autostainer, that perhaps can also show some problems. The fact, that we work with a machine, does not save us from failures. It can also be some human mistake, when the dilution was not prepared correctly.
In my opinion staining results should be comparable from run to run. Especially when the pretreatment (fixation, cutting) is in the same range.
Hmmm..... the slides were all prepared at the same time by the same person and I am comparing the same cuts with different antibodies...
I am using a Dako auto stainer, all the solutions come ready but the DAB and the antibody dilution.
I also think the results should be the same, specially because I need the control to be the same in order to be able to trust my results.
Since this is not the first time it happens I also take special care in the preparation of the solutions, but I have run out of ideas on what could be happening. Thank you for taking the time to answer ! : )
I recommend to ask the technical service of Dako.
uneven spread of reagens on the slides, issue with dispensed volumina, issue with quality of the kit, different results within the same run or different runs....? and so on
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