TUNEL labeling in Histochoice-fixed sections results in a high number of false positives (i.e. every nucleus is positive). Omitting tdt from the reaction prevents any labeling. When we use the same protocol in formalin-fixed samples we do not have the same false positive issue. We have tried a 1min microwave step with samples in 0.1M Citrate Buffer, pH 6.0, and have tried with and without saponin pre-treatment. Any suggestions about reducing non-specific labeling are welcome, but we are particularly interested in hearing from those who have used Histochoice fixative. Thank you!!
When you say you are getting 'false-positives', what exactly do you mean?
In normal tissues you would not expect to see cells that are TUNEL positive, as apoptotic cells are not at all common. Slides containing 5 μm–thick formalinfixed
paraffin-embedded slices of rat mammary gland on day 1 and day 7 of involution can be purchased from Zyagen (San Diego, CA, USA). This tissue remodeling is by far the finest model system of apoptosis and should serve as a positive control.
If you do TUNEL correctly you will see labeling in cell nuclei and in the cytosol. Nuclear DNA always contains some steady state 3'OH DNA ends and mitochondrial DNA even more.
Thanks for your response. Typically in a healthy heart, we see very few apoptotic nuclei. This is definitely the case in our formalin-fixed sections. However when we use the same protocol with our Histochoice-fixed sections, EVERY nucleus is labeled (literally every cell). This is the case even in our vehicle-treated samples.
What you observing is the normal wear and tear of steady state DNA damage; you are seeing background DNA damage and high resolution x60-x100 will allow you to see the mtDNA damage.
Since you're not inducing the same degree of crosslinking with Histochoice as with formalin, your problem is probably over-permeabilising your cells. I would try a milder permeabilisation protocol and/or eliminating the permeabilisation step with a DNase positive control to verify that TdT is able to reach nick ends.