Life Technologies (Molecular Probes) has a kit called Backdrop for live cells but not sure if it will work with fixed cells.They also have signal enhancers that might be useful. Do you have a positive control that you can use to make sure what you are trying to detect is present in the post-mortem brain? Sometimes if there is no signal you will only see background. Is there a different technique - for example IPs or Western Blots - that can be used to make sure what you are trying to detect is present in the brain tissues?

http://products.invitrogen.com/ivgn/product/R37603

Do you have autofluorescence present in your tissue when no secondary antibodies are present? Also, are you using TSA amplification on your primary antibody? If so, it is important to quench your tissue with peroxidase inhibitors before proceeding with staining.

Try adding Sudan Black at the end of the staining protocol: it should reduce autofluorescence, even though it will quench your signal a bit too.

Let me recap your question.

It seems that you are doing a fluorescence detection experiment with a fluorophore-labeled secondary antibody in a tissue section that contains also red-blood cells in your area of of rest. You excite fluorescence in "green" and detect through a bandfilter in the "green-to-yellow" spectral band. Autofluorescence is a "natural by product" of excitation with light from almost the whole visible and near infrared region. It results from hemoglobin and derivatives of hemoglobin in aging samples. It is difficult to get rid of, since there can be so much of it. A suggestion could be to rinse samples before fixation to get rid of as much as possible RB's.

You could do Na-Borohydride treatment and then staining with antibody

Thanks to all for your suggestion. I will try with a treatment with peroxidase inhibitor (hydrogen peroxide) before post fix and staining. Because my tissues are already fixed in PFA, I hope this treatment is effective. Sudan black reduce the intensity of my stainings and I do not use it. I will try also with Ns-Borohydride as suggested by Maria. I will let you know.

We have seen autofluorescence in mouse red cells both in FACS and fluorescence microscopy. It was useful there to help identify mouse blood vessels - see our publication at Stem Cell Research & Therapy 07/2012; 3(4):23. (Barclay et al), uploaded in Research Gate. However I have not seen extensive human red cell autofluorescence in FACS or in microscopy in unfixed or PFA-fixed (paraffin embedded sections) in the methods we used. You can check our published methods there to see if your differ. I do recall from somewhere that trypan blue is a fluorescence quencher - but it would also quench your antibody fluorescence. Maybe it would work as a pre-treatment??

(0.0l M

Tris±HCl and 830 mg NH4Cl/100 ml) is used to lyse red blood cells, I guess u could treat the sections with this buffer before fixation.

Please check out if this information is useful. reference is given below.

( We use this buffer to remove erythrocyte contamination by lysing it while isolating lymphocytes)

http://download.thelancet.com/flatcontentassets/H1N1-flu/immunity/immunity-4.pdf

Many thans Rahul. unfortunately I can not treat tissue before fixation, before are autoptic tissue already fixed by the Tissue Bank. PFA-fixed blocks arrive in my lab. Moreover, this procedure is very useful for snap frozen tissue!

I will let you know.

B

Originally it is meant to reduce endogenous peroxydase activity, but in some very bloody tissue we have used a preincubation with 3% hydrogen peroxide in methanol for 30 minutes of the FFPE sections for FISH. It will react with RBC and will destroy most of them resulting in a significant quenching.

Another option is the use lanthanides and time-resolved microscopy.

see here some publications:

http://www.ncbi.nlm.nih.gov/pubmed?term=Time-resolved%20microscopy%20%20lanthanide

Good luck,

Karoly

Hello Barbara,

I have a similar sort of problem. Did any of the suggestions work for you?

Jonathan

Hello Jonathan. No, at the moment. I tried to incubate my sections with hydrogen peroxide before Immunofluorescence staing, as suggested, to quench peroxidase activity and reduce autofluorescence, but after this treatment the immunostaining was completely negative! All immunoreactivity was lost.

I will try other methods and I will let you know.

Is it possible to give the section a wash with ionic liquid 1-butyl-3-trimethylsilylimidazolium hexafluorophosphate (BtmsimPF6). The author of the paper says it selectively removed hemoglobin. possibly if the quenching is due to hemoglobin from red blood cells it should be selectively removed by above ionic liquid.

http://www.sciencedirect.com/science/article/pii/S0039914008000714

Selective extraction/isolation of hemoglobin with ionic liquid 1-butyl-3-trimethylsilylimidazolium hexafluorophosphate (BtmsimPF6)

De-Hong Cheng, Xu-Wei Chen, Yang Shu, Jian-Hua Wang,

Research Center for Analytical Sciences, Box 332, Northeastern University, Shenyang 110004, China

I'm having the same issue. I treated my sections with h2o2 but the RBC are still there. My boss has suggested me to do a perfusion. We are working with mouse heart. I have not try the perfusion. I will like to try the Ionic 1-butyl-3trimethylsilylimidazolium hexafluorophosphate (BtmsimPF6) as suggested by Rahul before the perfusion. Ms. Barbara please let me know if any other method works for you. Thank you everyone!

Dear Yoanna, if you work with animal models the perfusion is the best way. When I worked with mice I always perfused mouse in toto and I have never had this problem with the tissues. Now, for autoptic brain tissue the problem of the red blood cells exists, mainly for the PFA-fixed tissue blocks.

In this paper: http://www.ncbi.nlm.nih.gov/pubmed/18585212 is indicated the method of BtmsimPF6 to extract hemoglobin, but it is indicated that this method is effective on human whole blood without any pretreatment. So, I'm afraid of this treatment it could be indicated for unfixed tissues but not for PFA-fixed tissue, that are those I'm used.

I agree with Cees Otto. I have the same problem. We are working with mouse heart ( aorta). In the green channel we can see tons of RBC. I discussed with the doctor I work for. He suggested me a perfusion.

The fluorescence is not due to background staining - mouse red cells are AUTO-fluorescent in red & green channels at 488 excitation (human red cells are not). The only way is to remove the red cells, or quench the (auto) fluorescence - by pretreatment (the quencher must be then removed or it will quench the fluorescent conjugates on antibodies used for subsequent staining). I seem to recall someone suggesting that pretreatment with trypan blue might work as a quencher.

Hi Barbara, I am having similar problems with erythrocytes in my FFPE tissues. Did any of the suggested methods worked at the end?

Thanks!

Hi Barbara..Help!!! do you have any solution for the problem? Thanks

Hi,

i suggest some protocols. One of them is to use 3% H2O2 with PBS or TBS, not methanol, because the methanol could destroy the fluorescece. The other way is the comercial solution Eliminator (http://www.merckmillipore.com/NO/en/product/,MM_NF-2160). I work with human brains and i have a lot of problem with autofluorescece. Other possible solution is the sudan black, for this reagent you can find some protocols in internet.

Best

I experienced a protocol that substantially reduces autofluorescence in my brain sections: I treat for 10 minutes my sections after the Immunofluorescence procedures with a solution of 10 mM CuSO4 in 50mM NH4Cl, final pH = 5. Then I wash with DDH2O and seal sections with my standard mounting medium. The results are very nice!

Hi Barbara,

do you do this treatment before or after nuclear staining (Dapi, Hoechst)?

Thanks!

Under your current situation H202 can help. But 3% is a bit high, we use 0.3% in PBS for 30mins, this significantly reduces RBC autoflouresences 

All good suggestions.

My lab has used the following to reduce or eliminate auto fluorescence:

• 0.3M Glycine dissolved in PBS or PBTx (PBS with 0.1% triton-X)
• 0.1% Sudan Black (powder dissolved in 70% ethanol)

Glycine was used before blocking and applying the primary antibody.

Sudan black was used after the primary, but before the secondary.

I've attached the protocol for you to see as a reference. This was used for human skin, so it may not be directly applicable to your work without modification.

All the best and cheers,

Paul

 Hi Paul Gradie

is there any deference if we use Sudan black after secondaries incubation?

I haven't checked, but I doubt it would be better. Auto-florescence isn't influence by the secondary (but of course non-specific signal is). You'll just have to give it a try and post the results.

Thank you very much everyone for your feedback. At what point during the staining process would I apply the hydrogen peroxide to the slide? 

Very old question but I have found post-fixing slides in 4% PFA (pH 7.4) for 10 min at room temp completely removes red blood cell autofluorescence. I do this at the beginning of the protocol but I think it would work at the end also.

Jack is this incubation of 4%PFA you do after deparaffinisation  of  Paraffin embedded tissue section on slide? 

Also Barbara thanks for this post got lot of info I facing exact same problem I hope you have solution by now if yes please let me know. Thank you. 

Hi All,

Just discovered this thread and is similar to what I am trying to solve. I am using IHC-P and having trouble with endogenous peroxidase within the erythrocytes of my tissue. I am a second year student on placement and so do not have access to fresh tissue to remove the blood cells via perfusion or through pre-fixation treatment. So I was wondering is there anything I can incubate my slides with that will destroy the red blood cells? I have tried a range of H2O2 concentrations from 3%-0.3% from 15 minutes to half an hour and I am still getting noise within my negative control (primary omitted). I am trying to stain a membrane protein so cannot use triton x etc as it will destroy my membranes.  I am coming to the end of my placement and my results currently are only a progression through optimisation so quite frustrating. Any help would be greatly appreciated.

Thank you,

Josef.

Thank you very much everyone for the interest and participation in the technical issue that afflicts many researchers  working with human tissues, especially tissue from the brain.

I would wish to respond everyone but this is a bit difficult. But everyone have added new information and suggestions based on his/her own experience.

In my lab I’m working and have worked with a lot of tissues subjected to different fixation treatments and, in my experience, I found that each fixation protocol generates  different problems in autofluorescence that require different treatments to reduce it.

Red blood cells autofluorescence is present always but mostly in sections derived from PFA-fixed blocks (during autopsy, tissue was removed and fixed in PFA4% for several days before freezing at -80 °C. Protocols for this is available, if of interest), or in formalin-embedded tissue blocks. The problem of red blood cell auto fluorescence is lower in snap frozen tissue blocks.

After reading a lot of interesting suggestions, now I treated my sections before the Immunofluorescence procedures with a solution of 10 mM CuSO4 in 50mM NH4Cl, final pH = 5 for 10 minutes.  Then I washed 3 times with DDH2O and then subjected my sections to the immunofluorescence procedure.  This reduced significantly auto-fluorescence in sections from paraffin embedded tissue.

 I agree with Jack Barrington: incubation with 4% PFA for 7-10 minutes before staining works well on snap frozen brain tissue. The autofluorescence of red blood cells is significantly reduced.  But this protocol was not efficient on PFA-fixed frozen brain tissue, perhaps because tissue blocks were already fixed in PFA. 

 Thank you Paul Gradie for the glycine/Sudan black protocol.  I tried with 0.1M Glycine in PBS, pH 7.4 for 5-10 min, but the result was not satisfactory. I also tried with Sudan black procedure after the immunofluorescence procedure but I observed that the intensity of the specific signals was also reduced. So, I will try your complete protocol and I will let you know.

 Joaquin Gonzalez Fuentes suggests the use of H2O2 (I have not tried this yet in fluo), a treatment that I use for the immuno-peroxidase protocol.

 A colleague suggests that we maybe can quench the autofluorescence by exposition to UV-light prior to staining with the labelled antibody. At least it reduces autofluorescence of macrophages and erythrocytes. He write.” I put on the uv-light in our bench and place the horizontally lying slides covered with buffer to avoid drying - e.g. pbs - and expose them 30 to 60 minutes to the UV-light. I tried it with brain, spleen and liver. The background/autofluorescence was distinctly diminished”. Thank you very much, this procedure is very interesting and I will test it as soon as possible on my fixed-frozen sections and I will let you know.

Let's stay in touch for future developments.

A new product just released from Vector Laboratories works well to remove autofluorescence due to red blood cells in FFPE. The product is called TrueVIEW. Good luck!

Thank you for all of the helpful tips. I work with bovine and porcine ovarian sections and erythrocyte AF is a major problem for me. I will try the UV exposure, hydrogen peroxide and copper sulfate/ ammonia chloride protocols to try to reduce this and let you know what worked best for me.

Thanks for the interesting tips, as this is always a challenge with non-perfused FFPE tissues. I've been solving it more at the imaging stage-if I want to count my signal, I use a far-red secondary for detecting my protein of choice, then collect images of the autofluorescence in the green channel as well. This way I can eliminate the highly green-positive autofluorescent signal in the erythrocytes, while counting the pure, isolated far-red signal. Obviously this is not a perfect solution, but helpful in some circumstances. Sudan black only eliminates lipofuscins and Sodium borohydride is only useful for aldehyde background, so I haven't found a perfect erythrocyte answer yet-looking forward to trying the other methods listed here!

Cannot recommend TrueVIEW enough. It worked perfectly for RBC autofluorescence in murine brain sections. See attachment.

Hi Jack, Can you tell us your exact protocol? We've been testing all of these recommendations in placental tissue from guinea pigs and have found the CuSO4 and UV worked better than Sudan Black, True View or True Black methods. In our hands True View wasn't that impressive

https://vectorlabs.com/trueview-autofluorescence-quenching-kit.html

Looks promising will let you know how it work in my hands.

Hi Karen, I followed the standard protocol (but modified to re-image). Briefly:

I stained slides and imaged them, then floated off the coverslips before draining excess and applying the trueview (pre-mixed in the specified order) for 5 minutes. I then washed 5 x with PBS, added mounting medium, coverslipped and re-imaged immediately.

Couple of things to note which may explain the discrepancies though. First, I am working with 20 um murine brain that has been fixed and cryoprotected. Second, the RBC autofluorescence was the result ofheat-induced antigen retrieval, for which I do not know how it occurs and how Trueview quenches, but I do know that UV does not work at all.

Thanks Jack!

Sudan Black B at 0.1% worked great for me when incubated with my slides for 10 minutes prior to antibody or blocking buffer addition.