I have been experiencing this problem while IHC-p staining my brain tissues. Staining is frequently uneven with gradient-like staining pattern (around 50% of my slides are affected). This happens with different monoclonal antibodies (apart from this staining single cells like CDs). All staining solutions (primary, secondary ab, enhancer, etc) cover my slides in excess, and the entire tissue is exposed to them. They do not leak. I use standard antigen retrieval protocol (citrate buffer, 30 minutes at 97 C) and my sealed slide boxes almost completely submerged into 97 C water bath during antigen retrieval (so there is no temperature differences at different parts of the glass slide). I found that it is almost impossible for me to achieve even staining pattern in the entire tissue.
If the absence of staining is consistently in the same locations within a tissue, this could possibly be an issue of incomplete fixation of the tissue before embedding in paraffin. For fixation in 10% neutral-buffered formalin, this is a common issue if tissues are too thick or not fixed in a large enough volume of fixative.
Otherwise, you might also see a gradient pattern of staining absence if deparaffinization and rehydration are incomplete prior to antigen retrieval and antibody staining.
Dear Arseniy Yuzhalin ,
In agreement with Jayne Wiarda , I can add and recommend staining the same slides with general staining days such as H-E or toluidine blue to test fixation problem or section detachment during the antigen retrieval process. In my experience, section thickness, antigen retrieval (heat), slide brand (commercially charged or hand made), and also fixation during sampling are the most important causes of inconsistent staining in IHC.
Good luck,
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