Hi,

I have a question for unbiased quantification method for tumor section staining.

I am currently doing TUNEL assay on primary breast tumor (Triple negative breast cancers) sections from mice. I have notice that the quantification data is very sensitive to where I take an image of.

In my case, the size of primary tumors were more than 400mm3 when they were collected. And all the tumors, regardless of sham or treatment groups, had varying degrees of necrotic or apoptotic core, which may be attributed to restricted oxygen and nutrition transport?

1.I assume that you should exclude those apoptotic or necrotic core tissues? Then, can I assume that event of necrotic or apoptotic core in primary cancer is completely independent of treatment (chemotherapy)?

2. For TUNEL assay, I observe the TUNEL staining of necrotic tissues that does not colocalize with DAPI staining. And these necrotic tissues, outside of core necrotic tissue, are more frequently observed in drug -treatment group. Would you still consider including those regions without DAPI colocalization?

If anybody give me general guideline for quantification of TUNEL staining or IHC staining, it would be great. I would like to get an experts opinion and be consistent and unbiased in quantification of primary tumor tissue staining.

Thanks!!