We are performing multiplexed immunofluorescence on formalin-fixed, paraffin embedded Hodgkin lymphoma. A major issue has been autofluorescence or non-specific binding of what we think are eosinophils, especially in the Texas red and GFP channels.
We have tried Sudan Black with little success.
Do you have any ideas on how to minimise the background without compromising the other signals we want?
Try to image a Sample without your Staining, at first. This will help you to decide if it is really unspecific binding or Background. When it is unspecific binding - well wash wash wash; block block block.
When it is background.... Take Images in every channel you have on your microscope. In some Tissues the Autoflourescence is less in the far-red Channels. When this is the case consider labeling with Cy5 or even Cy5.5, Cy7 or similar (Alexa680, Alewxa 750, Atto 700...).
You could also try to use a Confocal or Spinning-disc-Confocal to only collect your light from the plane you are imaging in - should also reduce Background.
I do not have any helpful tips to reduce autoflourescence in tissue by chemicals. Everything I tried before did not help or reduced also the signal massively. There are some Papers out there describing how to clarify tissue, but I never tried.
Formalin triggers autofluorescence. Try e.g. glutaraldehyde for fixation. Non labeled specimen can be used to study autofluorescence properties as suggested above. I found Glycin (200 mM) to reduce autofluorescence in formalin fixed tissues. Clearing does not affect autofluorescence properties in the first sense. Good luck
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