Did you try to reduce autofluorescence using glycine in PBS?

Make sure all of your blocking solutions are fresh and free of bacteria. Often, bacterial contamination will result in high amounts of autofluorescence. Are these paraffin embedded sections? If so, you will need to remove the wax before incubating in the citrate buffer (at high temperature). Give some more details of your staining process and perhaps we can nail down the culprit of the AF.

Hello

1-prepare tissue sections as usual.

2-At the usual blocking step, block with serum (from same species as the secondary antibody) for 30 min at room temperature.

3-Wash 3 X 2 min with PBS Tween 20. Incubate sections with an unconjugated affinity purified F(ab) fragment anti-mouse IgG (H+L) for 1 hr at room temperature, or overnight at 4°C. Try this blocking antibody at 0.1 mg/ml although the optimal concentration will need to be assessed by the end user.

Proceed with antibody staining.

Also, you can decrease background through :

1- Incubate sections with 1% Triton (in PBS) at room temperature for 30 min to 'clean' the tissue.

Or

2- Use TBS-Tween 20 as a washing buffer, this often gives less background than using PBS-Tween 20.

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http://media.cellsignal.com/www/pdfs/resources/product-literature/application-if-brochure.pdf

http://www.sciencegateway.org/protocols/pdf/ihc_icc_protocol_guide.pdf

Good Luck

I completely agree with the above answers.

In addition, try to serial dilution of the antibody you used to choice best staining with less background.

good luck

Elliot Glotfelty - The buffers are fresh. My sections are paraffin embedded. Before the incubation in the citrate buffer I keep them in 60C for 2 hours and wash in xylene solution to remove the wax.

Saleh Alkarim - I usually use 1% Triton diluted in TBS instead of PBS. It doesn't help a lot.

I am almost agree with the above answer.

Few more suggestion....

1. Before Fixation treat the sample with Signal enhancer.(https://www.thermofisher.com/order/catalog/product/I36933).

2.Treat with 3% hydrogen peroxide for 15 min.

2. Block with serum(10%) for 2 hr.

3. Dilute the antibody in standardized ratio in Blocking buffer.(https://www.thermofisher.com/order/catalog/product/37520?SID=srch-srp-37520).

4. Use Background suppressor after each staining.(https://www.thermofisher.com/order/catalog/product/B10512?SID=srch-srp-B10512).

Autofluorescence can be reduced very nicely using sodium borohydride washing, where, after rehydrating and permeabilizing the section, you wash 3 x 10 minutes with VERY freshly made (immediately prior to putting on the section) 1 mg/ml sodium borohydride in PBS. You'll see it fizzing on the section. Then wash it off and continue with blocking.

Also, if these are paraffin sections, which are always worse for autofluorescence than cryosections, I have found that deparaffinizing in limonene solvent (like Histoclear) greatly reduces autofluorescence compared to using xylene.

Subhadeep suggested the Signal Enhancer (which I helped develop), but it does not reduce autofluorescence. Rather, it prevents non-specific binding of dyes due to dye charge (where negatively-charged dyes will bind to positively-charged areas on cells and tissues). I highly recommend using it with all fluorescent antibody labeling, for that reason.

He also suggests use of BackDrop background suppressor, but that is really meant for quenching signal of media and dyes outside of live cells, in the green range, not for antibody labeling of fixed and permed samples.

Try to avoid 488 (green) secondary antibodies, as most of the autofluorescent signals lies there.

Some people recommend incubation for few minutes in Sudan Black (SBB). You can find more information in this paper

Article Benefits and Pitfalls of Secondary Antibodies: Why Choosing ...

Most of the important things are alreadsy mentioned. Here just some recommandations:

1. Don't rinse between blocking and primary antibody step, because otherwise the effect of blocking gets lost.

2. If you are working with primary abs which come from the same species than your tissue you will see no autofluorescence, you will see unspecific backgraund staining. To avoid this either use high concentrated normal sera, min 20% for blocking as well as for the primary ab raction, or try to find a suitable ab from another species.

3. If you can't distinguish between specific singnal and unspecific background and you are just looking for one protein I recommand to use the steptavidin/ABC- Peroxidase DAB staining. I tmakes it much easier to find out weather your result are specific or not, especially if you run additionaly a negative control.

Antigen retrieval with heat produces more autofluorescence than using enzyme digestion. Far red conjugates (e.g. AF647) will show less autofluorescence than red, green (AF488) is the worst.

Aside from all the above excellent ideas, I suggest to change the fluorophore: move to the longer wavelengths, even to infrared. At this range tissues have very little auto fluorescence.

Thank you for all your replies. I used SBB buffer and also the glicyne solution. Additionaly I use AF594 instead of AF488. The results are quite good, much better than before.

Dear Krystof,

using paraformaldehyd you always get some autofluorescence. We usually use a different kind of fixation. Bouin´s solution works better in our hands.

Best regards,

Daniela