I am observing autofluorescence in my formalin fixed paraffin embedded skin samples.
I see a bright signal using the DAPI channel after rehydration before blocking and this is carried over into the FITC channel after staining with Alexa 488 secondary alone. I do not know what is causing this, therefore unable to choose the right quenching agent. Has anyone encountered this before? If so what helped clear the autofluorescence to get a more specific staining pattern. My options for quenching now are Sodium Borohydride/ Sudan black or Sunlight/UV for photobleaching. I have attached a word doc with some reference images and the staining protocol I am following now.
Suggestions are welcome.
your protocol do not describe melting of paraffin, it start from deparaffinization. you are getting strong background due to paraffin (which is the biggest problem for FFPE). melt paraffin at 80 degree for 30 min then transfer immediately into xylene, shake occasionally. this will help a lot to reduce autofluorescence. and later add either sudan black or max block autofluorescence reducing reagent. it shall work according to my experience.
I did not know about melting the paraffin at 80 deg. I will try out both your suggestions. Thank you very much!
Right now I am using A594 and A647 secondaries and using confocal microscopy to separate out autofluoresence and overlap. It has been working very well but this obviously has its limitations. Is the max block from Biolynx?
max block autofluorescence reducing reagent i am using is from dianova. it have solution A for reducing background and solution B for enhancing fluorescent signals.
Vector Laboratories offers a product called TrueVIEW Autofluorescence Quenching Kit that is based on technology very different from Sudan Black, and may help with your application.
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