I'm going to use Lysotracker for frozen sections to assess autophagy. Now I have some questions about it.
1. I read someone's work indicating that Lysotracker Red but not Green can be used for tissues that need fixation. Is this true? Anyone knows the reason?
2. From the paper I read, they stained the tissue with LysoTracker first, and then fixed it. But if I used the frozen sections that have already been fixed, will the LysoTracker work?
3. Can I do the LC3 staining at the same time? If anyone has the experience, should I incubate the tissues with Lysotracker and LC3 Ab at the same time or should I do either one first?
Thanks.
Dear Yinghui,
LysoTracker dye is a cell permeable dye which stains acidic organelles in live cells. Generally, it can't be used in already fixed tissues as fixation with alcohol or aldehydes changes pH gradient required to stain acidic compartments.
However, some LysoTracker stains can be used for staining the tissues that need fixation but in case fixation can be done ONLY AFTER staining.
To label lysosomes in fixed tissues you can do Immunolabeling for LAMP1 / LAMP2/ cathepsin B which can also be used with LC3 staining.
Best,
Ritu
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