Although most people use a reporter like GFP-LC3, I have immunostained autophagosomes with the LC3B antibody from cell signaling technology on paraffin- embedded mouse tissues after antigen retrieval.

I advice the use of LC3 antibodies from nanotools (works both in IF and paraffin embedded section, please see Belaid et al. Cancer Res 2013, Chargui et al Tox Sci 2011). Best wishes

Thanks colleagues, but immunostaining is not suitable for my tasks. I plan to use my sample for laser microdissection with following microarray analysis so, my impact have to be very gentle.

Thank you Joshua for your detailed answer.

But do you have an experience of positive autophagosome staining with AO or MDC in fixed and wax-embedded cells? In my hands these dyes work just as vital dyes (in the case of autophagosome staining).

Same to you and many thanks, Joshua!

I have used LC3 antibodies to stain for autophagosomes. Novus biologicals makes a good one.

Thanks

I think that AO is useful only when the cell is alive (for autophagosome staining). I tried to stain fixed samples with MDC but result was very poor because of high autofluorescence. But I work with Arabidopsis may be you’ll have success with spleen.

Hi Dmytro,

I was following this question answer. I am having similar type of problem. I am using LysoTracker Green to detect AO. First I tried arabidopsis leaf with no success. I used confocal microscopy. Later I tried root. Now, I can see some signals but I am not sure what am I looking at because people usually published images with hundreds of AO structure, I am having may be 3/4. Could you solve the problem? I will be glad if you reply me. Bests