I am trying to isolate Alzheimer disease specific plaques from frozen brain sections using indirect immunofluorescence. Does anyone know a good solvent/reagent/buffer that can initially solubilize the surrounding tissue around the plaques in order to isolate these plaques alone and then solubilize these insoluble plaques? Does anyone have any other good plaque isolation technique suggestion?
Additionally, are 70% formic and 2% SDS the only good solubilization buffers used to solubilize insoluble amyloid-beta components?
Dear Iswariya
What do you want to do after you have isolated the plaques? If you want to run mass spec or HPLC, I believe it is better to leave the tissue untreated.
Laser microdissection is very useful when isolating plaques in tissue. With the technique, you are able to visualize the plaques and then draw a 'circle' around the plaque with the laser. The plaque is then removed by air pressure or flow into an eppendorf tube.
Take a look at this recent publication:
Acta Neuropathol. 2013 Jan;125(1):133-44. doi: 10.1007/s00401-012-1041-1.
They use laser microdissection to dissect plaques and then analyze them using various techniques.
I hope this helps, although I wasn't able to answer your specific question.
Santi
Dear Santi
Thank you for your suggestions.
Yes, mass spec is one of the techniques I would use to characterize the plaques components. However, the whole tissue is giving a lot of non-specific proteins (• Homeodomain protein OTX2, • Phosphatase methylesterase 1, • Tubulin beta-4A chain isoform 3 etc.). Therefore, I am looking for a technique which would enhance or separate the plaque components alone.
Laser microdissection is my last option (based on the availability of the microscope, resources etc). I have already read in detail the technique in: Curr Protoc Cell Biol. 2009 Sep; Chapter 3:Unit 3.33 3.33.1-33. doi: 10.1002/0471143030.cb0333s44. Therefore, I am trying other basic techniques before I opt for this.
Hi Iswariya
I've been trying the same thing with different ultra-centrifuge protocols and the one that gave me better results at the beginning was the protocol 2 in Curr Protoc Cell Biol. 2009 Sept, Chapter 3:Unit 3.33 3.33.1-33. doi: 10.1002/0471143030.cb0333s44. I need to modified some parts of it for my purposes, but the first try we did with this one was OK. Have you tried any modifications to this protocol?
Javiera
Hi Javiera
I have still not tried the Sucrose grad centrifugation, but have been trying to modify the protocol 1 for using in IP procedures. But, the Formic extracts are destroying the immune complexes/protein G beads and hence, no antigen is being pulled out with these beads (despite the neurtralization step with Tris base). And other milder buffers will not solubilize Amyloid beta complexes.
Did the sucrose gradient work well for you? Which species tissue did you use and what volume?
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