Dear Mostafa,

You can wash your samples with an Nacl solutions 0.05M or NaBr 0.2M for 5 min 3 times, yess I know this protocol seem strange but actually I used it  during my PhD to reduce the autofluorecence of Chitosan scaffolds and it works very well.

Best.

Philippe Bouaziz

The autofluorescence can be caused by quite a big number of natural fluorophores (most common are collagen or elastin for example), so it is I think impossible to completely remove it. Suppressing is possible, for example elastin emission can be shifted to the red spectrum using Pontamine sky blue dye (but honestly I've never used it, just know that it is possible). This will not however remove the autofluorescence completely; if you're using fluorescent labels, the best solution would probably be to use another label that won't cause a crosstalk with the autofluorescence.

Dear Philippe Bouaziz:

Thank you for your help. I will try to do it and will see how it works. :-)

Dear Martin Cepa:

Thank you for your help. Actually I do not want eliminate all autofluorescence. Just wanna try to remove as mush as I can till I see my cells and virus(FITC"cells" and PE"virus").

Hi Mostafa,

What equipament do you use to your fluorescence analysis? If you use confocal microscopy you could remove some of this autofluorescence.

Let me know more about it.

Dear Joao Paulo Rodrigues Marques:

Thank you. Yes, I am using confocal microscopy. May I know which specific information do you need?

Dear Mostadfa,

First of all, It is interesting to know the model of the confocal microscopy that you are using. Here in Brazil I have the acess to Confocal Laser Leica TCS SPE. I know that there is possibility to allow the detector to recive specific wavelenght. So, if you analize your sample first without your  virus(FITC"cells" and PE"virus", and then you can see what wavelenght you can detect. So, after that you can put your sample (virus) and make the detector avoid the wavelenght produced by autofluorescence.

In other others,  you identify fluorescence emission spectra and remove cross-talk between detection channels.

Please see the attached file and take a look of how to remoeve cross-talk.

I hope this information helps you. Good luck!

Dear Mostafa,

I also had many problems with autofluorescence, and specially with formaldehyde fixed tissues.

Have a look at the following papers, I tried the different protocols, and using Sudan Black worked best for me. It also varies a lot from tissues, as there are different endogenous molecules which can be autofluorescent.

http://www.ncbi.nlm.nih.gov/pubmed/11724904

http://www.ncbi.nlm.nih.gov/pubmed/17548270

Good luck!

Dear Mostafa,

I agree with Aura, and others, you have to try out varies recipes ( in the above papers) to find one which works for your problem. Yet, thinking of chicken duodenum, there is also the possibility of tissue contaminated with the highly fluorescent e.g. tetracycline-anitbiotics, which may even accumulate, or you may immobilize fluorescent gut content during fixation. I would make sure that your guts are well rinsed before fixation - and if you suspect that you may have tetracyclines etc. you may try to add appropriate solvents during the dehydration procedure. for paraffin embedding.

good luck

Werner

Dear Joao Paulo Rodrigues Marques ,

I have access to Olymous(IX81, fv1000 confocal) and Zeiss(LSM5 poscal confocal microscope). I tried before for my samples without virus(PE)also,but still I have got background. 

Thank you for your word file, bur i do not know why I could not open it?!

Regards,

Mostafa

Dear Aura Ileana Moreno-Vega,

Thank you for these papers. I will go through the papers and see how can solve the problem.

Regards,

Mostafa 

Dear Werner Baschong ,

Thank you for your  advice. I will try also this time like that and will be more careful to see what will be outcome. I hope it will work also.

Regards,

Mostafa

Dear Philippe Bouaziz,

Thank you for your answer to my question about how reduce fluorescent . I want to know that when I should wash the samples with Nacl solutions 0.05M or NaBr 0.2M for 5 min 3 times? before I put in PFA or after that ?

" 1st I wash as you said before and then put in PFA? OR put in PFA and then wash it? "

Regards,

Mostafa

Hi,

In the end, chemical methods of eliminating autofluorescence do not work well. They might reduce the AF some, but some of them also reduce wanted signal, leaving you in the same situation you were in. And you will be extremely lucky if you happen to find a single emission channel that is not affected by autofluorescence.

The only certain way to deal with autofluorescence is to use a multispectral imaging system designed to unmix, or separate, autofluorescence from your signal(s) of interest. You can find more information about those in these papers:

http://www.ncbi.nlm.nih.gov/pubmed/16969820

http://www.ncbi.nlm.nih.gov/pubmed/18972383

If you have any further questions about it, please don't hesitate to contact me!

Jim

Dear James Robert Mansfield,

Thank you for your help and your papers. I will read these and I hope my problem will solve. I will contact you if face any problem. Thank you for your attention to this matter

Regards,

Mostafa

Dear James Robert Mansfield,

I could not download the article"Visualization of Microscopy-Based Spectral Imaging Data from Multi-Label Tissue Sections". Is it possible for you give me a link which I can download?

Regards,

Mostafa

Dear Mostafa, I certainly agree with JR Mansfield, spectral imaging & linear unmixing is the most reliable way to tell apart wanted and unwanted signal. This works nicely, even at the sub cellular level, and avoids to make erroneous statements, because the organelle you are talking about is not the organelle you think it is..

http://www.ncbi.nlm.nih.gov/pubmed/17416619

http://www.ncbi.nlm.nih.gov/pubmed/16568270

Afterwards, things get complicated, because there is no such thing as autofluorescence, it is usually a mix of different endogenous fluorophores with different tissular and sub cellular distribution. Depending on you spatial resolution (and the spectral band you are imaging in) different components will dominate. And many of them (NAD, flavins, lipofucsin, but also - for cultured cells - internalised phenol red from the cell culture medium) have broad and indistinct spectra, both on the excitation and emission side. (see e.g., the pure AF figure in the recent overview

http://www.ncbi.nlm.nih.gov/pubmed/24681159).

Careful filter design helps (often the common "GFP cube" is NOT the best filter set in YOUR very experimental scenario, and many companies offer the fabrication of custom band-pass filters that might be better suited to your experiment. It's a bit more expensive but not that much.

A sometimes underrated contributor to autofluorescence is the microscope: coverslip, immersion oil and the objective itself, but as you seem to work in tissue, this might not apply to your experiment.

With the advent of more and more sensitive detectors, a general rule is that all material becomes fluorescent at some level of sensitivity (particularly with short-wavelength excitation, e.g., 405 nm) and the only way to be aware of that are proper controls...

Here is a link from which you can download the publication:

https://www.researchgate.net/publication/23439993_Visualization_of_microscopy-based_spectral_imaging_data_from_multi-label_tissue_sections

Article Visualization of Microscopy-Based Spectral Imaging Data from...

Thank you for all your kinds. I have to try to find which one will work for my research.

Thank you