Question

Do you have any suggestion to eliminate red blood cells autofluorescence present in PFA-fixed sections?

Second antibodies I use are routinely used in my lab, give no aspecific stainings on other cell types and we already tried with diffent dilution, but the problem persists! I stain sections from post-mortem human brain.

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  • Domina Falcone · McMaster University
    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
  • Tony Lim · McGill University
    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.
  • Alessandro Bitto · Drexel University College of Medicine
    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.
  • Cees Otto · Universiteit Twente
    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.
  • Maria Nemethova · Austrian Academy of Sciences
    You could do Na-Borohydride treatment and then staining with antibody
  • Barbara Serafini · Istituto Superiore di Sanità
    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.
  • George Barclay · The University of Edinburgh
    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??
  • Rahul Raghavan .TV · University of Calicut
    (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
  • Barbara Serafini · Istituto Superiore di Sanità
    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
  • Karoly Szuhai · Leiden University Medical Centre
    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
  • Jonathan Wilson · University of Porto
    Hello Barbara,
    I have a similar sort of problem. Did any of the suggestions work for you?
    Jonathan
  • Barbara Serafini · Istituto Superiore di Sanità
    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.
  • Rahul Raghavan .TV · University of Calicut
    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
  • Yoanna Bello · Sanford-Burnham Medical Research Institute
    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!
  • Barbara Serafini · Istituto Superiore di Sanità
    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.
  • Yoanna Bello · Sanford-Burnham Medical Research Institute
    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.
  • Kotarkonda Lakshmi kanth · Justus-Liebig-Universität Gießen
    Block with 4 % bovine serum albumin, it will reduce back ground staining overall, also in RBC, before incubating with primary antibody.
  • George Barclay · The University of Edinburgh
    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.

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