We usually choose pH6 in DAKO PT Link module but sometimes the signal is too low. When we try the same samples with pH9 the signal is much better but we fear that this is just an image of a false positive.
How can we be sure the pH chosen is the correct and the image is reliable?
Below you will find some more general and hopefully useful recommendations:
* There are reports of false positive stainings using different pretreatment methods (not only high pH buffers), but if you perform appropriate controls, there is no need to be afraid of this rare phenomenon. I suspect some people overemphasize this scary phantom to make IHC look lmore complicated than it is.
* Think out of the box, there is a number of really nice pretreatment buffers beyond what Dako offers that work well in the PT-Module. The typical two lane choice of "only" regarding pH6 vs pH9 is typical Dako education and is generally not bad, but there is more than one way to skin the cat.
* There are at least 4 different HIER buffers, that work well in waterbath/steamer/PT-Modules: CITRATE (pH6), EDTA (pH8), Tris-EDTA (pH9), TRIS (pH10). Generally speaking: the higher the pH, the stronger the staining, the higher the potential damage to the morphology. I like to use pH8, if citrate does not give enough signal, that frequently does the job. Sometimes a short pH10 pretreatment can give fantastic results.
* I am not a fan of enzymatic pretreatment, eventhough there are many examples for really good results. The reason I do not like enzymes is that generally the results are less reproducible than HIER (for obvious reasons). There are some antigens though (example: pan-cytokeratin, clone MNF116), that give significantly better results with enzyme. If using enzymes I would only use RTU and IVD certified ones, to have as little uncontrolable parameters as possible.
* A typical scheme for establishing a new antibody would be in my hands:
(I) 16 slides (8 controls, 8 questionable tissue) > (1) Citrate, pH6, (2) EDTA, pH8, (3) Standard RTU Enzyme, Proteinase K or Protease 25, (4) none and corresponding negative controls (ideally isotype controls).
(II) Choose best stained slide from (I) and perform titration series with best HIER method.
* One really good tip (see it as a reward for the amount of text above :-) ) : after HIER chill the hot slides immediately in cold washbuffer (4-8° C) and let them rest for 5-10 min. This improves the crispness of staining and the morphology.
Good luck with your experiments !
In order to be really sure, you need to try both pHs and use positive and negative control tissues. By that, I mean cells or tissues that you know will be immunoreactive and non-immunoreactive. If the differential between these cells/tissues looks good, you can have more confidence that the method is working well. Antigen retrieval is very antibody-dependent. Good luck!
In our lab we use 3 types of antigen retrieval: citrate (low pH), EDTA (high pH and protease.It is suggested that a new antibody should be tested with all of above forming a matrix with dilutions, unless data sheet helps. And to our experience, EDTA is superior to citrate most of time.
Below you will find some more general and hopefully useful recommendations:
* There are reports of false positive stainings using different pretreatment methods (not only high pH buffers), but if you perform appropriate controls, there is no need to be afraid of this rare phenomenon. I suspect some people overemphasize this scary phantom to make IHC look lmore complicated than it is.
* Think out of the box, there is a number of really nice pretreatment buffers beyond what Dako offers that work well in the PT-Module. The typical two lane choice of "only" regarding pH6 vs pH9 is typical Dako education and is generally not bad, but there is more than one way to skin the cat.
* There are at least 4 different HIER buffers, that work well in waterbath/steamer/PT-Modules: CITRATE (pH6), EDTA (pH8), Tris-EDTA (pH9), TRIS (pH10). Generally speaking: the higher the pH, the stronger the staining, the higher the potential damage to the morphology. I like to use pH8, if citrate does not give enough signal, that frequently does the job. Sometimes a short pH10 pretreatment can give fantastic results.
* I am not a fan of enzymatic pretreatment, eventhough there are many examples for really good results. The reason I do not like enzymes is that generally the results are less reproducible than HIER (for obvious reasons). There are some antigens though (example: pan-cytokeratin, clone MNF116), that give significantly better results with enzyme. If using enzymes I would only use RTU and IVD certified ones, to have as little uncontrolable parameters as possible.
* A typical scheme for establishing a new antibody would be in my hands:
(I) 16 slides (8 controls, 8 questionable tissue) > (1) Citrate, pH6, (2) EDTA, pH8, (3) Standard RTU Enzyme, Proteinase K or Protease 25, (4) none and corresponding negative controls (ideally isotype controls).
(II) Choose best stained slide from (I) and perform titration series with best HIER method.
* One really good tip (see it as a reward for the amount of text above :-) ) : after HIER chill the hot slides immediately in cold washbuffer (4-8° C) and let them rest for 5-10 min. This improves the crispness of staining and the morphology.
Good luck with your experiments !
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