Fwd: DAPI Staining Issue
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Hello Group,
Not a confocal microscopy question but a sample preparation question; I have a colleague who is getting very poor nuclear morphology from tumour xenografts that are fixed in 10% neutral buffered formalin for 24 hours, and then transferred to 70% ethanol. They are subsequently paraffin embedded and sectioned to 4um. The H and E stains also reflect poor nuclear morphology but it is most clearly evident when the sections are stained with 0.1ug/ml DAPI and mounted with Vectashield for widefield or confocal imaging. We are accustomed to seeing nuclei that are generally homogeneously stained, with a couple of nucleoli that are well defined. The problematic sections seem more vacuolar in appearance. These tumours do not have as many nucleoli as the image may suggest. Also, when stained with a marker that forms punctate foci within nuclei, the marker seems to accumulate on the edges of the more dense DAPI staining, rather than be distributed through the nucleus.
Any thoughts on this sample prep question would be greatly appreciated. I know there are many of you in this forum with expertise in tissue preparation. I have 2 images, one of good DAPI/nuclear morphology and one of poor; what is the convenient way let the group have a look at what I am trying to explain? Thanks very much to all. Cheers Farid -- Farid Jalali MSc Senior Research Technician- Lab Manager Applied Molecular Oncology/ Princess Margaret Hospital STTARR Innovation Facility/ Radiation Medicine Program Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Farid, Your description sounds like extraction or dislocation of cellular components by prolonged exposure to alcohol or by using wrong alcohol concentrations. Alcohol is a solvent for lipids and is able to affect the morphology of lipidic cellular components, typically membranes. The poor nuclear morphology evidenced with two different nuclear stains is a strong indication that your material is morphologically compromised. Try to work only on your best material and discard the compromised material. Regards Fulvio Fulvio Florenzano PhD Assistant Imaging Unit Lab. of Experimental Neurorehabilitation "S. Lucia" Foundation Via fosso del Fiorano, 64/65 00143 Rome Italy e-mail: [hidden email] lab phone: +39-6-501703080 Fax: +39-6-501703305 mobile: 333-2125293 -----Messaggio originale----- Da: Confocal Microscopy List [mailto:[hidden email]] Per conto di Farid Jalali Inviato: martedì 26 agosto 2008 3.42 A: [hidden email] Oggetto: Fwd: DAPI Staining Issue Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Group, Not a confocal microscopy question but a sample preparation question; I have a colleague who is getting very poor nuclear morphology from tumour xenografts that are fixed in 10% neutral buffered formalin for 24 hours, and then transferred to 70% ethanol. They are subsequently paraffin embedded and sectioned to 4um. The H and E stains also reflect poor nuclear morphology but it is most clearly evident when the sections are stained with 0.1ug/ml DAPI and mounted with Vectashield for widefield or confocal imaging. We are accustomed to seeing nuclei that are generally homogeneously stained, with a couple of nucleoli that are well defined. The problematic sections seem more vacuolar in appearance. These tumours do not have as many nucleoli as the image may suggest. Also, when stained with a marker that forms punctate foci within nuclei, the marker seems to accumulate on the edges of the more dense DAPI staining, rather than be distributed through the nucleus. Any thoughts on this sample prep question would be greatly appreciated. I know there are many of you in this forum with expertise in tissue preparation. I have 2 images, one of good DAPI/nuclear morphology and one of poor; what is the convenient way let the group have a look at what I am trying to explain? Thanks very much to all. Cheers Farid -- Farid Jalali MSc Senior Research Technician- Lab Manager Applied Molecular Oncology/ Princess Margaret Hospital STTARR Innovation Facility/ Radiation Medicine Program Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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In reply to this post by Farid Jalali
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Farid, This sounds like either a tissue viability issue or poor fixation. Does this morphology extend throughout the xenograft to the edges or is it more prevalent in the central regions? How big are the samples and are they simply immersion fixed? How is the tissue handled prior to fixation and is the formalin properly buffered? the other possibility that comes to mind is incomplete infiltration by the paraffin due to insufficient dehydration with absolute ethanol prior to the clearing steps. My US$0.02 (CA$0.015) Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ****************************************************************************** The box said "Requires WindowsXP or better", so I bought a Macintosh. ****************************************************************************** On Aug 25, 2008, at 6:41 PM, Farid Jalali wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Hello Group, > > Not a confocal microscopy question but a sample preparation > question; I have a colleague who is getting very poor nuclear > morphology from tumour xenografts that are fixed in 10% neutral > buffered formalin for 24 hours, and then transferred to 70% ethanol. > They are subsequently paraffin embedded and sectioned to 4um. The H > and E stains also reflect poor nuclear morphology but it is most > clearly evident when the sections are stained with 0.1ug/ml DAPI and > mounted with Vectashield for widefield or confocal imaging. We are > accustomed to seeing nuclei that are generally homogeneously > stained, with a couple of nucleoli that are well defined. The > problematic sections seem more vacuolar in appearance. These tumours > do not have as many nucleoli as the image may suggest. Also, when > stained with a marker that forms punctate foci within nuclei, the > marker seems to accumulate on the edges of the more dense DAPI > staining, rather than be distributed through the nucleus. > > Any thoughts on this sample prep question would be greatly > appreciated. I know there are many of you in this forum with > expertise in tissue preparation. I have 2 images, one of good DAPI/ > nuclear morphology and one of poor; what is the convenient way let > the group have a look at what I am trying to explain? Thanks very > much to all. > > Cheers > Farid > > -- > Farid Jalali MSc > Senior Research Technician- Lab Manager > Applied Molecular Oncology/ Princess Margaret Hospital > STTARR Innovation Facility/ Radiation Medicine Program > Toronto, Canada > 416-946-4501 X4351 (Princess Margaret Hospital) > 416-581-7754 STTARR at MaRS Building > 416-581-7791 STTARR Microscopy Suite |
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In reply to this post by Fulvio Florenzano
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We were always taught when preparing specimens for EM preparation to avoid long periods in low alcohol concentrations - 50% and 70% - as these would strip lipids esp. membranes and degrade the ultrastructure. Perhaps the same is happening here, you could try either keeping the specimens in formalin or in higher ethanol concentrations. Another alternative could be PBS-azide with a low BSA and storage in the fridge. Cheers Ray G -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Fulvio Florenzano Sent: Tuesday, 26 August 2008 7:38 p.m. To: [hidden email] Subject: R: DAPI Staining Issue Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Farid, Your description sounds like extraction or dislocation of cellular components by prolonged exposure to alcohol or by using wrong alcohol concentrations. Alcohol is a solvent for lipids and is able to affect the morphology of lipidic cellular components, typically membranes. The poor nuclear morphology evidenced with two different nuclear stains is a strong indication that your material is morphologically compromised. Try to work only on your best material and discard the compromised material. Regards Fulvio Fulvio Florenzano PhD Assistant Imaging Unit Lab. of Experimental Neurorehabilitation "S. Lucia" Foundation Via fosso del Fiorano, 64/65 00143 Rome Italy e-mail: [hidden email] lab phone: +39-6-501703080 Fax: +39-6-501703305 mobile: 333-2125293 -----Messaggio originale----- Da: Confocal Microscopy List [mailto:[hidden email]] Per conto di Farid Jalali Inviato: martedì 26 agosto 2008 3.42 A: [hidden email] Oggetto: Fwd: DAPI Staining Issue Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Group, Not a confocal microscopy question but a sample preparation question; I have a colleague who is getting very poor nuclear morphology from tumour xenografts that are fixed in 10% neutral buffered formalin for 24 hours, and then transferred to 70% ethanol. They are subsequently paraffin embedded and sectioned to 4um. The H and E stains also reflect poor nuclear morphology but it is most clearly evident when the sections are stained with 0.1ug/ml DAPI and mounted with Vectashield for widefield or confocal imaging. We are accustomed to seeing nuclei that are generally homogeneously stained, with a couple of nucleoli that are well defined. The problematic sections seem more vacuolar in appearance. These tumours do not have as many nucleoli as the image may suggest. Also, when stained with a marker that forms punctate foci within nuclei, the marker seems to accumulate on the edges of the more dense DAPI staining, rather than be distributed through the nucleus. Any thoughts on this sample prep question would be greatly appreciated. I know there are many of you in this forum with expertise in tissue preparation. I have 2 images, one of good DAPI/nuclear morphology and one of poor; what is the convenient way let the group have a look at what I am trying to explain? Thanks very much to all. Cheers Farid -- Farid Jalali MSc Senior Research Technician- Lab Manager Applied Molecular Oncology/ Princess Margaret Hospital STTARR Innovation Facility/ Radiation Medicine Program Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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In reply to this post by Glen MacDonald-2
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Farid
We recently did an indirect comparision of formalin/paraffin and paraformaldehyde/cryostat sections for nuclear morphology. Para had uniform morphology and did not show the extracted areas seen with formalin. Can you change your processing method? Dick Burry ----- Original Message ----- From: Glen MacDonald <[hidden email]> Date: Tuesday, August 26, 2008 12:52 pm Subject: Re: DAPI Staining Issue To: [hidden email] > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Farid, > This sounds like either a tissue viability issue or poor > fixation. > Does this morphology extend throughout the xenograft to the > edges or > is it more prevalent in the central regions? How big are > the samples > and are they simply immersion fixed? How is the tissue > handled prior > to fixation and is the formalin properly buffered? the > other > possibility that comes to mind is incomplete infiltration by > the > paraffin due to insufficient dehydration with absolute ethanol > prior > to the clearing steps. > > My US$0.02 (CA$0.015) > > Glen > > Glen MacDonald > Core for Communication Research > Virginia Merrill Bloedel Hearing Research Center > Box 357923 > University of Washington > Seattle, WA 98195-7923 USA > (206) 616-4156 > [hidden email] > > ****************************************************************************** > The box said "Requires WindowsXP or better", so I bought a Macintosh. > ****************************************************************************** > > > On Aug 25, 2008, at 6:41 PM, Farid Jalali wrote: > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello Group, > > > > Not a confocal microscopy question but a sample > preparation > > question; I have a colleague who is getting very poor > nuclear > > morphology from tumour xenografts that are fixed in 10% > neutral > > buffered formalin for 24 hours, and then transferred to 70% > ethanol. > > They are subsequently paraffin embedded and sectioned to 4um. > The H > > and E stains also reflect poor nuclear morphology but it is > most > > clearly evident when the sections are stained with 0.1ug/ml > DAPI and > > mounted with Vectashield for widefield or confocal imaging. We > are > > accustomed to seeing nuclei that are generally > homogeneously > > stained, with a couple of nucleoli that are well defined. > The > > problematic sections seem more vacuolar in appearance. These > tumours > > do not have as many nucleoli as the image may suggest. Also, > when > > stained with a marker that forms punctate foci within nuclei, > the > > marker seems to accumulate on the edges of the more dense > DAPI > > staining, rather than be distributed through the nucleus. > > > > Any thoughts on this sample prep question would be > greatly > > appreciated. I know there are many of you in this forum > with > > expertise in tissue preparation. I have 2 images, one of good > DAPI/ > > nuclear morphology and one of poor; what is the convenient way > let > > the group have a look at what I am trying to explain? Thanks > very > > much to all. > > > > Cheers > > Farid > > > > -- > > Farid Jalali MSc > > Senior Research Technician- Lab Manager > > Applied Molecular Oncology/ Princess Margaret Hospital > > STTARR Innovation Facility/ Radiation Medicine Program > > Toronto, Canada > > 416-946-4501 X4351 (Princess Margaret Hospital) > > 416-581-7754 STTARR at MaRS Building > > 416-581-7791 STTARR Microscopy Suite > > > -- > BEGIN-ANTISPAM-VOTING-LINKS > ------------------------------------------------------ > > Teach CanIt if this mail (ID 666264311) is spam: > Spam: > https://antispam.osu.edu/b.php?c=s&i=666264311&m=e2259132abf3Not > spam: https://antispam.osu.edu/b.php?c=n&i=666264311&m=e2259132abf3 > Forget vote: > https://antispam.osu.edu/b.php?c=f&i=666264311&m=e2259132abf3---- > -------------------------------------------------- > END-ANTISPAM-VOTING-LINKS > Richard W. Burry, Ph.D. Department of Neuroscience, College of Medicine Campus Microscopy and Imaging Facility, Director The Ohio State University Associate Editor, Journal of Histochemistry and Cytochemistry 277 Biomedical Research Tower 460 West Twelfth Avenue Columbus, Ohio 43210 Voice 614.292.2814 Cell 614.638.3345 Fax 614.247.8849 |
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Search the CONFOCAL archive at
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Farid, instead of vectashield, try aqueous solution of DAPI at around 50-100 microgram/mL in PBS, incubate for 15 min and wash briefly in PBS for 2-3 min and try. The problem might be the viscosity of the vectashield might hamper pernetration of the dye to the target in some cell or tissue types. Full penetration achieved while using aqueous solutions. This we reccommend to all samples when using DAPI. Hope this helps. Shiv At 08:49 AM 8/27/2008, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Farid Microscopy Facility Manager 8, Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 West Gregory Dr. Urbana, IL 61801 USA Office: 217.333.1214 Fax: 217.244.2496 [hidden email] http://core.igb.uiuc.edu |
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In reply to this post by Farid Jalali
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Farid, I agree with each of the responses to your question, which to summarize: 1) use paraformaldehyde made fresh which provides better more extended crosslinking because it remains a polymer for awhile until reverting to formaldehyde monomers. 2) proper dehydration is essential if using EtOH. This means ~70% EtOH, 80%, 90%, and finally absolute EtOH. This can still cause serious artifacts because of rearrangement of lipid membranes and extraction of some proteins and lipids. Without complete dehydration, the paraffin embedding can create apparent vacuolar structures. 3) Polylysine (PL) can promote dye sticking if the polylysine has not been fully blocked. The PF-amino binding is unstable unless the Schiff's base is reduced say with sodium borohydride or similar reducing agent. The polyamino groups can lead to electrostatic binding of negatively charged stains. This can be prevented by treating with fresh dilute (~ 1 mM) succinic anhydride or acetic anhydride before staining. 4) I have always had severe background staining using plastic coverslips with or without PL. Good for growing cells but their hydrophobicity really causes problems with a lot of stains because of the amphoteric nature of most dye molecules. I gave up on plastic decades ago. PL and 5 ug/ml fibronectin on coverslips attach cells for growing is good most the cell types I've used. 5) The H & E staining even if you are not doing the unfortunate paraffin embedding, will never give good nuclear DNA staining compared to DAPI, Hoechst, and Draq5. The two former stains are minor groove labels that work very well for nuclear DNA staining. Almost all other nucleic acid stains label all nucleic acids whether they are mitochondrial DNA, RNA, or any other _NA. I also agree that using a much higher DAPI concentration is sensible. Given the density of DNA in the nucleus you have to worry about saturating the DNA. Flow Cy. commonly uses 3 ug/ml which makes for reasonably quantitative analysis and growth stage evaluation using flow. I have to stop no, but feel free to email me the images you are having problems with. I am curious to see them. good luck, Mario [hidden email] [hidden email] [hidden email] >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Hello Group, > >Not a confocal microscopy question but a sample preparation >question; I have a colleague who is getting very poor nuclear >morphology from tumour xenografts that are fixed in 10% neutral >buffered formalin for 24 hours, and then transferred to 70% ethanol. >They are subsequently paraffin embedded and sectioned to 4um. The H >and E stains also reflect poor nuclear morphology but it is most >clearly evident when the sections are stained with 0.1ug/ml DAPI and >mounted with Vectashield for widefield or confocal imaging. We are >accustomed to seeing nuclei that are generally homogeneously >stained, with a couple of nucleoli that are well defined. The >problematic sections seem more vacuolar in appearance. These tumours >do not have as many nucleoli as the image may suggest. Also, when >stained with a marker that forms punctate foci within nuclei, the >marker seems to accumulate on the edges of the more dense DAPI >staining, rather than be distributed through the nucleus. > >Any thoughts on this sample prep question would be greatly >appreciated. I know there are many of you in this forum with >expertise in tissue preparation. I have 2 images, one of good >DAPI/nuclear morphology and one of poor; what is the convenient way >let the group have a look at what I am trying to explain? Thanks >very much to all. > >Cheers >Farid > >-- >Farid Jalali MSc >Senior Research Technician- Lab Manager >Applied Molecular Oncology/ Princess Margaret Hospital >STTARR Innovation Facility/ Radiation Medicine Program >Toronto, Canada >416-946-4501 X4351 (Princess Margaret Hospital) >416-581-7754 STTARR at MaRS Building >416-581-7791 STTARR Microscopy Suite |
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In reply to this post by Farid Jalali
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Howdy Group,
Thanks so much to all for excellent suggestions and comments. I will work with my colleague to sort through the comments and our procedures to figure out what we can do better or different. Best to all. Farid -- Farid Jalali MSc Senior Research Technician- Lab Manager Applied Molecular Oncology/ Princess Margaret Hospital STTARR Innovation Facility/ Radiation Medicine Program Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Farid, I think that the processing of the tissue is correct. I wonder that how the xenografts were produced. DAPI signal appeared as punctate foci within nuclei and accumulated on the edges of nuclei Is the sign of apoptosis or necrosis Did the investigator inject the cells into the animal with metrigel or other reagents to keep the cells alive? If not, ischemic necrosis will happen in the center of tumor. The another problem was that how the tumor was harvested. Did they fixed the tissue right away? Those problems can solved by checking the histology of HE stained section. Mei Ling At 08:43 PM 8/27/08, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Howdy Group, >Thanks so much to all for excellent suggestions and comments. I will work >with my colleague to sort through the comments and our procedures to >figure out what we can do better or different. > >Best to all. >Farid > > >-- >Farid Jalali MSc >Senior Research Technician- Lab Manager >Applied Molecular Oncology/ Princess Margaret Hospital >STTARR Innovation Facility/ Radiation Medicine Program >Toronto, Canada >416-946-4501 X4351 (Princess Margaret Hospital) >416-581-7754 STTARR at MaRS Building >416-581-7791 STTARR Microscopy Suite |
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