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Need some Fluorescent Microscopy Guidance Please


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#1 wcsoccerfan

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Posted 29 August 2014 - 05:14 AM

Basic info:

 

Have OCT embedded lung sections (10uM, mouse, currently unfixed) at -80.  Need to do some staining.  Any advice would be welcome.

 

1)  We are a lab that has 60-70% of experiments ending in flow.  I'd like to use the same antibodies to do some staining of my sections, but most protocols involve secondary antibodies.  What are the odds my fluorophore conjugated primaries will work without any signal amp. from a secondary? Most of the primaries are monoclonal, FYI.  Probably depends on the antigen, but thought I would ask.

 

2)  I want to counterstain with DAPI, but all my proteins of interest are membrane bound proteins.  Thinking of fixing with 4% paraformaldehyde and not including a permableazation step.  Will DAPI still work without the perm step?

 

3)  I'm staining for CD31 (PECAM-1) and some lesser expressed proteins as well.  All of our CD31 primaries are fluorophore conjugated, and the other ABs are biotin conjugated.  I have the option of using a secondary AB or streptavidin-PE/FITC for those.  Which is the better option?  I would think the secondary for signal amplification, but if the biotin/streptavidin system has less background or something it might be better.  As a side note, I have great controls (sections from KO mice from the proteins I'm staining for) to compare staining to.

 

Thanks in advance, and I'll certainly take any other tips anyone has.


Edited by wcsoccerfan, 29 August 2014 - 05:42 AM.


#2 bob1

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Posted 30 August 2014 - 04:02 PM

1) as you say - it depends on the antibody. I have had good success with some directly conjugated, but generally go for secondaries, as it is cheaper and easier to get secondaries than trying to conjugate primaries.
 

2)DAPI might not work, but there are cell permeant alternatives, such as some of the Hoescht range of DNA dyes. PI should also work, but that will drop you the option of doing a red channel protein stain.

 

3)I would personally go for the secondary option. Biotin/streptavidin works too, but I have found it to be more difficult.






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