We are processing clinical samples of bone marrow smears, and have encountered autofluorescence that makes subsequent FISH and IHC virtually impossible. The problems are: 1. the samples are of whole bone marrow, not separated on Ficoll or any other gradient (that's something that won't be altered)
2. We want to preserve cell volume so any harsh treatment is out of question
What could we do to lower/eliminate autoflourescence in that case?
Thanks
A new product has been released from Vector Laboratories that reduces autofluorescence from many sources in FFPE sections. The product is called TrueVIEW. It has been shown to be very effective for fixative induced fluorescence and also fluorescence due to collagen, elastin, and red blood cells.
esp. for quenching or blocking free ‘aldehydes’ after fixation (cf. e.g. ROTH J. et al.(1981): Enhancement of structural preservation and ICC staining in low temperature embedded pancreatic Tissue, J.Histochem.Cytochem.29,663-671) NH4Cl(ammonium Chloride)
50 mM NH4Cl in 0.1M -0.13M (phosphate) buffer (or in PBS or TBS as outlined already by my foreposters) for at least 5-10 mins will be used.
As other possibilities (besides lysine, glycine, NH4Cl, Na-borohydride, etc.) regarding quenching agents in the literature there have been mentioned also (no exhaustive listing!):
sulphorhodamine (cf. e.g.:
STAUGHTON TJ, McGillicuddy CJ, Weinberg PD.(2001): . J.Micr. 201 / #1, 70-76, 2001)
use of appropriate dyes
(e.g. sections are stained with an appropriate dye which, for example, quenches the broad band autofluorescence, like Toluidine Blue for GFP, methylene blue, trypan blue,
cf.: REVIEW: , by Nicholas Billinton and Andrew W. Knight, in Analytical Biochemistry 291, 175–197 (2001), doi:10.1006/abio.2000.5006, available online at http://www.idealibrary.com)
as well as e.g. use of
Sudan Black B (e. g. cf.: Research Article - , by Yanfei Yang and Ali Honaramooz, in: Anatomy Research International, © Hindawi Publishing Corporation Vol. 2012, Article ID 820120, 10 pages, doi:10.1155/2012/820120.
If you would like to read more about the matter “quenching-blocking-removing-reducing autofluorescence and mechanisms “ you naturally could search in RG too:
just insert the following URL-addresses in your RG-browser-field (you’ll get requests and answers with the same or similar background as your request):
https://www.researchgate.net/post/How_to_overcome_strong_autofluorescence_on_paraffin_sections
Established Means of Lowering Autofluorescence
Nonetheless, it is expected that the increase in fluorescence seen in FFPE tissue is due to the generation of C=C or C=N bonds. To address this problem, sodium borohydride has been utilized in several studies to chemically reduce the C=C and C=N bonds and diminish the autofluorescence. Borohydride and similar reducing agents, however, are unstable at neutral pH. Whereas researchers have noted some success when using sodium borohydride to lower tissue autofluorescence, the procedure is not reproducible and the reagent is difficult to work with.
An additional method that has been investigated to diminish tissue autofluorescence is photobleaching. When this technique is used, tissue sections are exposed to high-intensity UV radiation for long periods of time to irreversibly photo-oxidize the fluorescent tissue elements. Photobleaching, which is often used in conjunction with other treatments, has been shown to be somewhat effective; however, it is time consuming. Also, photobleaching requires equipment that most immunohistochemistry laboratories lack.
Historically, the main method that has been employed to lower tissue autofluorescence has been to treat the tissue with solutions of Sudan Black or similar nonfluorescent diazo dyes. These hydrophobic dye molecules will generally bind nonspecifically to tissue sections. After binding to the tissue, Sudan Black acts as a mask to lower the fluorescence through the absorption of incident radiation (dark quenching).
Specifically, Sudan Black is very effective at lowering the fluorescence due to lipofuscin in tissue. Lipofuscin is a brightly fluorescent pigment that accumulates with age in several tissue types. Lipofuscin granules are composed of lipid-containing residues of lysosomal digestion. Because of the hydrophobic nature of Sudan Black, it binds effectively to the lipid-rich lipofuscin granules and effectively masks their fluorescence. However, Sudan Black is less effective at lowering the fluorescence due to aldehyde fixation, connective tissue elements, and RBCs.
📷 https://www.genengnews.com/magazine/310/reducing-tissue-autofluorescence/
New Technology
A new method has been introduced that allows for the dramatic reduction of the autofluorescence due to formalin fixation, collagen, elastin, and RBCs. This innovative technology, sold as the Vector TrueVIEW Autofluorescence Quenching Kit, involves the treatment of tissue sections with an aqueous solution of a hydrophilic molecule that binds electrostatically to collagen, elastin, and RBCs. This nonfluorescent, negatively charged molecule also binds effectively to formalin-fixed tissue including colon, pancreas, prostate, tonsil, spleen, kidney, gallbladder, and thymus.
Once bound, the TrueVIEW Quencher significantly lowers the fluorescence of tissue components. It is likely that this reagent lowers autofluorescence through a combination of static and dark quenching mechanisms.
Because the TrueVIEW Quencher is hydrophilic, the treatment of tissue sections is performed in an aqueous buffer, and does not require a 70% ethanol pretreatment step that is necessary with Sudan Black procedures. In addition, the TrueVIEW Quencher treatment requires only a two-minute step at the end of the immunofluorescence assay. Further, TrueVIEW Quencher is compatible with common fluorophores such as fluorescein, Alexa Fluors, DyLight fluors, cyanine fluors, and green fluorescent protein.
Hope this helps you and others anyway,
best wishes,
A new product has been released from Vector Laboratories that reduces autofluorescence from many sources in FFPE sections. The product is called TrueVIEW. It has been shown to be very effective for fixative induced fluorescence and also fluorescence due to collagen, elastin, and red blood cells.
esp. for quenching or blocking free ‘aldehydes’ after fixation (cf. e.g. ROTH J. et al.(1981): Enhancement of structural preservation and ICC staining in low temperature embedded pancreatic Tissue, J.Histochem.Cytochem.29,663-671) NH4Cl(ammonium Chloride)
50 mM NH4Cl in 0.1M -0.13M (phosphate) buffer (or in PBS or TBS as outlined already by my foreposters) for at least 5-10 mins will be used.
As other possibilities (besides lysine, glycine, NH4Cl, Na-borohydride, etc.) regarding quenching agents in the literature there have been mentioned also (no exhaustive listing!):
sulphorhodamine (cf. e.g.:
STAUGHTON TJ, McGillicuddy CJ, Weinberg PD.(2001): . J.Micr. 201 / #1, 70-76, 2001)
use of appropriate dyes
(e.g. sections are stained with an appropriate dye which, for example, quenches the broad band autofluorescence, like Toluidine Blue for GFP, methylene blue, trypan blue,
cf.: REVIEW: , by Nicholas Billinton and Andrew W. Knight, in Analytical Biochemistry 291, 175–197 (2001), doi:10.1006/abio.2000.5006, available online at http://www.idealibrary.com)
as well as e.g. use of
Sudan Black B (e. g. cf.: Research Article - , by Yanfei Yang and Ali Honaramooz, in: Anatomy Research International, © Hindawi Publishing Corporation Vol. 2012, Article ID 820120, 10 pages, doi:10.1155/2012/820120.
If you would like to read more about the matter “quenching-blocking-removing-reducing autofluorescence and mechanisms “ you naturally could search in RG too:
just insert the following URL-addresses in your RG-browser-field (you’ll get requests and answers with the same or similar background as your request):
https://www.researchgate.net/post/How_to_overcome_strong_autofluorescence_on_paraffin_sections
Established Means of Lowering Autofluorescence
Nonetheless, it is expected that the increase in fluorescence seen in FFPE tissue is due to the generation of C=C or C=N bonds. To address this problem, sodium borohydride has been utilized in several studies to chemically reduce the C=C and C=N bonds and diminish the autofluorescence. Borohydride and similar reducing agents, however, are unstable at neutral pH. Whereas researchers have noted some success when using sodium borohydride to lower tissue autofluorescence, the procedure is not reproducible and the reagent is difficult to work with.
An additional method that has been investigated to diminish tissue autofluorescence is photobleaching. When this technique is used, tissue sections are exposed to high-intensity UV radiation for long periods of time to irreversibly photo-oxidize the fluorescent tissue elements. Photobleaching, which is often used in conjunction with other treatments, has been shown to be somewhat effective; however, it is time consuming. Also, photobleaching requires equipment that most immunohistochemistry laboratories lack.
Historically, the main method that has been employed to lower tissue autofluorescence has been to treat the tissue with solutions of Sudan Black or similar nonfluorescent diazo dyes. These hydrophobic dye molecules will generally bind nonspecifically to tissue sections. After binding to the tissue, Sudan Black acts as a mask to lower the fluorescence through the absorption of incident radiation (dark quenching).
Specifically, Sudan Black is very effective at lowering the fluorescence due to lipofuscin in tissue. Lipofuscin is a brightly fluorescent pigment that accumulates with age in several tissue types. Lipofuscin granules are composed of lipid-containing residues of lysosomal digestion. Because of the hydrophobic nature of Sudan Black, it binds effectively to the lipid-rich lipofuscin granules and effectively masks their fluorescence. However, Sudan Black is less effective at lowering the fluorescence due to aldehyde fixation, connective tissue elements, and RBCs.
📷 https://www.genengnews.com/magazine/310/reducing-tissue-autofluorescence/
New Technology
A new method has been introduced that allows for the dramatic reduction of the autofluorescence due to formalin fixation, collagen, elastin, and RBCs. This innovative technology, sold as the Vector TrueVIEW Autofluorescence Quenching Kit, involves the treatment of tissue sections with an aqueous solution of a hydrophilic molecule that binds electrostatically to collagen, elastin, and RBCs. This nonfluorescent, negatively charged molecule also binds effectively to formalin-fixed tissue including colon, pancreas, prostate, tonsil, spleen, kidney, gallbladder, and thymus.
Once bound, the TrueVIEW Quencher significantly lowers the fluorescence of tissue components. It is likely that this reagent lowers autofluorescence through a combination of static and dark quenching mechanisms.
Because the TrueVIEW Quencher is hydrophilic, the treatment of tissue sections is performed in an aqueous buffer, and does not require a 70% ethanol pretreatment step that is necessary with Sudan Black procedures. In addition, the TrueVIEW Quencher treatment requires only a two-minute step at the end of the immunofluorescence assay. Further, TrueVIEW Quencher is compatible with common fluorophores such as fluorescein, Alexa Fluors, DyLight fluors, cyanine fluors, and green fluorescent protein.
Hope this helps you and others anyway,
best wishes,
I would like to recommend using the Vector TrueVIEW Autofluorescence Quenching Kit. It reduce the autofluorescence due to formalin fixation, collagen, elastin, and RBCs.
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