VIT University
Question
Asked 4th Jan, 2016
Is this lipofuscin autofluorescence in my FFPE sections?
I am working on a double IF protocol in FFPE sections of whole mouse hind paws, and below is an image of the IgG control in the 488 & 594 channels (secondaries), as well as a combined image with DAPI. After some research, I'm thinking the positive signals look most similar to lipofuscin autofluorescence, but wanted to verify since it seems to be more of a problem neural/renal tissues. I believe Sudan black treatment in EtOH would be the right course of action to quench it? I'd really like to resolve this so that I can be more confident identifying positive signal in my experimental sections. Thanks for your time!
Most recent answer
Yes, 0.3% Sudan black treatment, amonium ethanol treatment and use of Cy5 or Cy7 may help you to understand your problem and answer your queries.
All Answers (7)
University of Antwerp
Hi Jessica,
I have previously performed IF on cryosections from neuronal tissues and was faced with lipofuscin in neuronal cells. The pattern in your picture does not really resemble what I saw. In my case it was a more granular fluorescent signal in the cytoplasm, while in your case it seems more like a fluorescence blob. I am of course not familiar with hind paw tissue, so maybe someone else has experience with this staining pattern.
Have you checked the tissue without the secondary? To see whether it is tissue autofluorescence or a secondary antibody problem..
What I can say is that Sudan B in EtOH indeed is a good option for reducing lipofuscin. But be careful as over treatment can also mask your specific signal.
Hope this helps (at least a bit) :)
Beijing Normal University
I agree with Samuel. At least in the mouse brain, it is like this. But I rarely saw lipofuscin autofluorescent.
University of Pavia
Obviously depending of tissues and cells involved. the morphology of intracellular lipofuscin deposits seem more finely granular rather than so massive bodies. In any caseYour images seem compatible with lipofuscin autofluorescence. Among emission and excitation wavelengths compatible with lipofuscin, may be useful the paper:
Spectral profiling of autofluorescence associated with lipofuscin ...
UNSW Sydney
'...sections of whole mouse hind paw...' -- The endogenous fluorescence could be from a lot of things (elastin? collagen? pigmented cells? fixation artifact? hair?) and its hard to diagnose without more information regarding what we're looking at, the details of your protocol, etc..
My general advice would be to try a variety of treatments in parallel, including imaging unfixed slices and sodium borohydride treated slices (to see if fixation is an issue) before any labeling. Start there and then add in complexity.
The two nuclear options are (1) put enough primary/secondary on to be considerably brighter than autofluorescence, or (2) expose to very bright light for some time before labeling (added link to 'A multispectral LED array for the reduction of background autofluorescence in brain tissue' for a newer take on this method). If one of these works, note explicitly what you did and how the controls look in your eventual methods section. Best of luck!
-edit- I just realized you were noting the images were collected following labeling in secondary-only controls, so maybe the signal is your secondaries. Unlikely, but in this case you need to wash more to allow passive diffusion of secondary out of some sticky areas or treat more to push down non-specific binding.
University College London
Hi Jess-
I work on the retina of the eye where there is a lot of autofluorescence emission from photoreceptor outer segments and retinal pigment epithelium. On top of that, Macrophages clearing lipofuscin debri will also autofluoresce. Whenever in doubt we have the following solutions:
1) Lipofuscin is combination of a number of lipids, hence it has a a broad ex/em spectrum:
A) You can use secondary antibodies with a longer wavelength emission spectrum than lipofuscin - Cy5, or Cy7, for instance.
B) If you are using a widefield fluorescence microscope with a broadband led white light source (such as <http://goo.gl/D2Nz2n>) lipofuscin will emit a broadband orange-yellowish indigenous fluorescence easily distinguishable from the ‘spiky’ emission of your green, or red secondary.
2) Alternatively, you can use a heavy metal stain to avoid the issue of autofluorescence altogether (see: ‘Nitro blue tetrazolium staining: a morphological demonstration of superoxide in the rat retina’)
All the best and a Happy New Year.
-Peter
Medical University of Bialystok
These are erythrocytes (for 99.9% ;). Bigger magnification would show it better.
Similar questions and discussions
How good is Qeios as an academic journal?
This weekend, I decided to accept an invitation to review a paper by a new journal called Qeios. It is a journal without an editor, but I learnt that it is controlled by AI rather than traditional humans as journal editors/editorial assistants. It also supports Open Science and open review methods.
It appears that Qeios utilises AI to find out the best reviewers from databases across the world. This gets new people to review, and these people are always related to the topic, and are mostly experts! This is an example of AI being harnessed for good!
As an author, I have not published here but as a reviewer, it is my first review feedback that has been posted or reviews in #Qeios journal.
From my initial finding, these Qeios papers are basically preprints, which means that the authors can receive about 10 comments to improve the quality of the submission. That does not mean it will be accepted for final publication.
Although, the paper also gets a DOI, then it gets indexed on google scholar! You can find my first review for the journal online, at https://www.qeios.com/read/CLC992 for the paper's preprint which has DOI: https://doi.org/10.32388/CLC992
Their papers can be searched on Google and some scholars as well as academic experts have already endorsed #Qeios papers. What about you? Will you publish in it? Will you review for the journal?Does it look like it will overtake traditional journals? What are their advantages and disadvantages?
Related Publications
This staining was performed to detect fetuin-A in activated microglia. CD68 is a marker, that stains activated microglia, macrophages and monocytes. Fetuin-A and microglia were detected in paraffin sections (1 μm thickness) of formalin-fixed human brain tissue. CD68 was stained using a monoclonal mouse-anti-human antibody (Dako Cat# M0814, RRID:AB_...
Expression of EGFP-positive cells in the gastric groove. Immunofluorescence staining of stomach cryosections. EGFP-positive cells clustered in the gastric groove and were positive for ChAT and the α-subunit of the G-protein gustducin. Nuclei were counterstained with DAPI. Squares indicate enlarged areas. Scale bars: 50 μm.
Immunofluorescence Staining of Sohlh1
Sections of 4-d-old testes were used. Nuclei were counterstained with DAPI (blue).
(876 KB PPT)