I have attached an image of a mouse brain section (the mouse is transgenic and generates human amyloid) stained with Congo red and viewed under polarised light. The result is blue birefringence. The literature says apple-green/green/green-yellow birefringence in these conditions is diagnostic for amyloid. Can it be said for certain this is amyloid?
All I have been able to turn up so far when reading the literature is that the polariser and analyser may not have been at right angles to each other, but the images were taken by a technician that should have operated the setup correctly. What might cause blue birefringence of Congo red?
Edit: the thumbnail appears to have a green haze but the birefringent material is definitely blue, or even blue-white.
Tried to reupload a larger version of the image but can't seem to re-add it to the original question, have attached it to this comment.
Congo red is placed in a chiral environment upon binding to amyloid. Thus excitation and emission from the the of the excited state take place in an anisotropic environment, which allows propagation of the light at different speed, thus splitting of the beam is observed: http://en.wikipedia.org/wiki/Birefringence. This birefringent staining of amyloid is more specific than the staining with Thioflavin S or -T. Congo red binds to other organic material too, oligomeric carbohydrates (as fund in starch). Yet birefringency is usually not observed in these cases.
If you want to be shure to have stained amyloid use Ab directed antibodies or more recent amyloid specific stains such as MeXO4 our dye 4090 (J.Med.Chem 2012).
You may apply spectral deconvolution (if you have access to the necessary detector) to differentiate form lipofuscin fluorescence.
http://pubs.acs.org/doi/abs/10.1021/jm300653b
Hi Jason,
Would not say this looks like typical amyloid, but rather like Lipofuscine dots within the neuronal layer of the hippocampus (I suppose). Aß1-42 injections into the hippcoampus can look like this, but those are not typical amyloid plaques.
Are you so lucky to have a fluorescent exciation on the microscope? CongeRed on amyloid (and Tangles in humans - see attachment) emits wellin the red spectrum thus a green excitation might show you the fluorescence and you don't need to rely on the birefringens.
Doctor Do you think going to make any differences what's kind of color makes amyloid?
Amyloid has nothing to do with Alzheimer Diseases.
Jason,
Since you didn't do the work yourself it is difficult to say what this is. Almost anything will fluoresce when excited with blue light but you can't get blue fluorescence when excited with red light. It just doesn't work that way. I believe the absorbance for CR is 540 nm..green light. My guess is your technician had the wrong excitation filter in there.
Warren
These do not seem to have stained amyloids in the tissue. I surmise the interference of other components during processing/staining steps. I have faced similar problems with salts/sugars giving different colors under cross-polarizer.
Perhaps you can find an explanation in this paper: doi:10.1038/labinvest.3700714.
Good luck!
Jason, Warren is right. Sometimes you can do the excitation at less energetic wavelengths and see them by some back-transfer related mechanisms at more energetic ones but normally it doesn't work that way. I would advise you to use specific antibodies for Abeta amyloids for that staining. Recently I have published an Immunohistochemistry study with some benzothiazol compounds that could help you with your research (Martins et al, ACS Med. Chem. Lett., 2013, pp 436–440).
Andre
When stained by a conventional HE what is the morphology of this material? Amyloid appears as pale amorphous eoisinophillic which is difficult to identify in brain but it may be possible. If the tissue is cut at the correct thickness, your microscope is set up correctly, (which as other people have commented on is extremely important), and your positive control Congo red stained slides shows green birefringence under polarised light, then this material isn’t amyloid. It could be a number of other crystalline structures that demonstrate birefringence under crossed polarised light. Janet
@ Ian - nuclei were stained with haematoxylin.
@ Gjumrakch - amyloid plaques are part of the Alzheimer's disease diagnostic criteria - see http://www.ncbi.nlm.nih.gov/pubmed/22265587
@Warren - I'm not sure I understand, my understanding of polarised light microscopy is that you use two polarising filters on a brightfield microscope. Where do excitation filters come in? I've done traditional epifluorescence on the section and not observed red fluorescence (using texas red excitation filter).
@Glenys - 1. I used the Puchtler method, dye supplied by Sigma 2. I am unsure, all I know is the microscope is fully dedicated to polarised light microscopy 3. We didn't have time to get anymore than a few pics of the test samples on the day, so no pics of positive control 4. The tissue is formalin fixed, cut on a cryostat at 20 um. I am aware this is too thick for optimal visualisation of birefringence but unfortunately the tissue was cut long before we decided to do Congo red.
@Glenys I started a thread and project here to create a sort of centralized place to share protocols. If you want, feel free to add anything
https://www.researchgate.net/post/Amyloid_protocols_project
@Ian I think this is the paper you're looking for but I don't think its whats going on here
Label-Free, Quantitative Assay of Amyloid Fibril Growth Based on Intrinsic Fluorescence
http://onlinelibrary.wiley.com/doi/10.1002/cbic.201300103/full
Do anyone can tell me why the polarized light and Congo Red stain of this BALB/c kidney is not apple green bi-refringence in the glomerular tufts?
I attached the ppt file.
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