Hi Almudena,

I never had any back-ground problems with blocking steps.

You can easily test by incubating a tissue section with your blocking-agent only and see if you get any signal besides the normal autofluorescence.

with kind regards,

Ronald

Hi Almudena,

   Could you provide a little more information about your experiment (tissue, how it was fixed, what other antibodies you might be trying to multiplex with, are you trying to do sequential labeling or are you labeling multiple antigens all at one time)?  My lab typically blocks before applying primary antibodies using 2-10% normal goat serum + 5% BSA + 0.5% triton X-100, which produces only low levels of background fluorescence in our tissue (retina and brain mostly). On the other hand, we have found that using normal Donkey serum in the blocking solution at these concentrations produces very high background. I don't know why. Other additional factors that affect the levels of fluorescent background you might need to consider include:

• The detergent can increase autofluorecence in areas that have high lipid content (like myelinated axons in the brain).
• Tissues that don't have immune privilege can produce high levels of non-specific labeling when trying to use a primary antibody raised in the same host as the tissue (i.e., using a rat primary antibody to label rat liver, etc.). The brain and retina have the blood-brain barrier to keep serum proteins and Igs out, but most other tissues (like liver) are bathed in serum proteins and Igs from the circulation, so the secondary anti-rat antibody will bind to the native Igs in the tissue and yield very high background. There are approaches for dealing with this and several companies sell kits for this type of labeling (mouse-on mouse kits, for instance).
• Autofluorescence can vary among fluorescence channel, with the blue and green channels typically showing higher levels than red or far red channels.
• If doing multiple immunolabeling, you could experience high levels of background and/or false-positive double labeling if the secondary antibodies cross-react with antibodies from other species. Use of well-characterized highly cross-adsorbed secondary antisera is advisable for multiple labeling.
• There are background fluorescence reduction procedures that have been described. My lab often uses a 1% NaBH4 pretreatment of our sections to help reduce autofluorescence (if you try this, you must make the solution using water- NaBH4 reacts violently with PBS and will wreck your sections).
• Fixatives can also produce high background fluorescence. Fresh paraformaldehyde usually produces low background levels, but formaldehyde that has been in solution for a long time can produce polymers that are highly fluorescent. Glutaraldehyde fixation produces very high fluorescence.

I hope some of this might be useful. Good luck.