Dear community, I have a set of tissue samples (N=179) stained using multiplexed IHC protocol (Opal 7plex kit). Samples were stained in batches ~ 14 samples per batch, manually. We had apprx. 12 staining procedures to process all samples. Further they were scanned using VectraPolaris platform (one protocol for all aqcuisitions). Multispectral images will be analyzed using inForm 2.4.2 software. I see quite a huge range of signal intencitities over the set of samples and wondering wheter the reason is staining procedure or the variability of tissue quality (breast cancer). From here is my question: do I need 1 specefic project in inForm for every indnvndual staining (ie for every 14 samples)? Or it is possible to use 1 protocol to run over all 179 samples? Or there are some methods to narrow down the number of required projects from 12 to 2-4, and group samples according to their intencity pattern profile? What criteria in that case have to be considered? As a read out we use tissue classification and cell phenotyping. Thank you.
In short: the optimal way is to use automated staining procedure, same exposure settings via aquisition and same protocol for batch analysis for all samples. So you have to deal only with intersample variability.
In some cases control and targed tissues could be placed on the same glass slide (as TMA).
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