Hi Richard, Spectral bleed through between channels is often a problem.

There are several approaches you can take

1) Titre the antibody-conjugate that is causing the problem downwards if the signal is too strong (i.e. to get the lowest amount of antibody that gives a good signal: noise ratio). You can check for spectral bleed through in the second channel in the absence of the other ab-conjugate (i.e. initially only use the HRP conjugate causing the problem). Using just the HRP will also show you if you are actually getting spectral bleed through in the other channels (the fact that it is in different compartments is indicative you are not)..

2) Analyse the section sequentially (i.e. probe with the CY3 conjugate by itself and record signal relative to control) then follow this with the HRP one (on the same section)..If you use coverslips the nail polish can be removed with a little acetone on a tissue (but do not allow section to dry out!).

3) I assume that the sections are 5-10 microns and you get several sections on a slide (I am assuming paraffin wax sections too). You could probe adjacent sections with each antibody separately and then image each and merge - I am not sure how you are staining but when I do sections I us a hydrophobic pen to draw around sections. The "hydrophobic barrier" allows you to use very small volumes of ab/stain/block etc.It also allows separate sections on one slide to be stained with different antibodies/dyes etc.

The other possibility (as you see the "signal" in other tissue compartments) is that what you are observing is not spectral bleed through or ab related but is in fact some type of auto-fluorescence. I assume you have a negative control section(s) (sections with no specific primary ab i.e. secondary only or with non-specific ab's)? If the problem is auto-fluorescence you should also see these signals in the affected channels in the controls. Note: auto-fluorescence in  IHC is generally seen outside of cells due to ECM components (especially elastin and collagen) however sometimes it can be observed inside cells as "punctate" spots which is generally due to i.e. lipofuscin.

Hope this helps!

Gary 

I am not sure if I understand your question correctly by "bleed through". But If you mean that there is blood in the area usually we isolate the tissue after perfusion with 4%PFA. This washes out any blood, so you do not have false positive signal.

The fact that the signal was in two different cellular comparts should in part ensure you about the quality of the staining. Anyway, if you are using a confocal microscope for the image acquisition, put the CY3 sequence first and make sure to avoid part of the 488 emission reducing your CY3 spectrum. If you are not using a confocal microscope, and the 488 staining is very strong, I am afraid that you can hardly avoid the bleed trough.

Hi Richard, Spectral bleed through between channels is often a problem.

There are several approaches you can take

1) Titre the antibody-conjugate that is causing the problem downwards if the signal is too strong (i.e. to get the lowest amount of antibody that gives a good signal: noise ratio). You can check for spectral bleed through in the second channel in the absence of the other ab-conjugate (i.e. initially only use the HRP conjugate causing the problem). Using just the HRP will also show you if you are actually getting spectral bleed through in the other channels (the fact that it is in different compartments is indicative you are not)..

2) Analyse the section sequentially (i.e. probe with the CY3 conjugate by itself and record signal relative to control) then follow this with the HRP one (on the same section)..If you use coverslips the nail polish can be removed with a little acetone on a tissue (but do not allow section to dry out!).

3) I assume that the sections are 5-10 microns and you get several sections on a slide (I am assuming paraffin wax sections too). You could probe adjacent sections with each antibody separately and then image each and merge - I am not sure how you are staining but when I do sections I us a hydrophobic pen to draw around sections. The "hydrophobic barrier" allows you to use very small volumes of ab/stain/block etc.It also allows separate sections on one slide to be stained with different antibodies/dyes etc.

The other possibility (as you see the "signal" in other tissue compartments) is that what you are observing is not spectral bleed through or ab related but is in fact some type of auto-fluorescence. I assume you have a negative control section(s) (sections with no specific primary ab i.e. secondary only or with non-specific ab's)? If the problem is auto-fluorescence you should also see these signals in the affected channels in the controls. Note: auto-fluorescence in  IHC is generally seen outside of cells due to ECM components (especially elastin and collagen) however sometimes it can be observed inside cells as "punctate" spots which is generally due to i.e. lipofuscin.

Hope this helps!

Gary 

When I say bleed through, I mean fluorescence between channels.  Sorry for being unclear.  The animals are perfused, there is no actual blood in the tissue.  As far as it being in 2 different compartments, it was not 100% clear.  It appears that it may be, but there is also some other cells that are positive that shouldn't be, which led me to initially believe that it was coming through both channels.  

The tissue is fixed in 4% PFA, and they are embedded in OCT.  My sections are usually 10-12 microns.  I will try these other suggestions.  Thank you.  

I would also pay attention to autofluorescence issues as mentioned above y Gary's last point. I encountered similar problems in the past -derived from lipofuscin auto-florescence in brain slices from adult mice- which were solved by CuSO4-based buffers.

Hello

For more details about IHC 

See attachment and links

http://web.qbi.uq.edu.au/microscopy/wp-content/uploads/2011/pdfs/QBI_HISTOLOGY_AND_MICROSCOPY_GUIDE.pdf

https://www.abdserotec.com/immunohistochemistry-antibodies-tips-and-tricks.html