Hello,
I have been performing some IHC staining on parafin embeded tissue.
All was good until recently.
In the latest slides I have prepared, when looking under the microscope, some areas need to be constantly refocused to properly see the side-by-side sections of tissue.
This did not happen in the workup experiments and I can not put my finger on what the problem might be.
The antigen retrieval, blocking, staining and mounting procedures were exactly the same since the begining. The tissue seems to be in good condition and I do not think there is a bubble causing this.
I am attaching example of the uneven focus issue.
Any idea on why this is happening is more than wellcome.
Thank you!
Hello,
May be, in this case the mounting media is not well spread over the section.
Try to clean the lenses of the camera also. Try to find another area of the specimen, without thick layer of mounting media between glasses.
And during focusing adjust the aperture of the camera manually at "infinity" point.
Regards!
Your tissue appears to be partially lifting from the slide, which is causing the uneven focus (tissue not on a flat plane). I would review which slide type you are using - are they good positively-charged slides ? Did you change the model since your previous experiments ? There can be significant differences in tissue adhesion with different models/brands of slides. Also your tissue appears to be quite fatty, which requires very good adhesion.
I would second Jean-Martin. But if it is only happening with this slide, then there is the potential that the mounting media is smeared over the coverslip. In this case, you should be able to clean the slip with xylene (or whatever solvent you used during dehydration and mounting).
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