[Histonet] Green Fluorescent Protein
Hi I am looking to demonstrate Green fluorescent protein in Para formaldehyde fixed, processed rat tissue. Apart from using an antibody against GFP, can the native GFP be detected? Gayle Callis, I believe you may be able to help me with this method. Thanks Melanie Melanie Black 082 469 3352 Cardiovascular Research Unit 3rd Floor; Chris Barnard Building Medical School; Observatory. 7925. University of Cape Town. South Africa. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Green Fluorescent Protein
The fluorescence itself will not be detectable, you would have to use an indirect means (like antibody, which you mentioned). That's just from my experience with it. Maybe Gayle Callis has a suggestion... Regards, Merced M Leiker Research Technician II Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. --On Tuesday, October 27, 2009 2:48 PM +0200 Melanie Black melanie.bl...@uct.ac.za wrote: Hi I am looking to demonstrate Green fluorescent protein in Para formaldehyde fixed, processed rat tissue. Apart from using an antibody against GFP, can the native GFP be detected? Gayle Callis, I believe you may be able to help me with this method. Thanks Melanie Melanie Black 082 469 3352 Cardiovascular Research Unit 3rd Floor; Chris Barnard Building Medical School; Observatory. 7925. University of Cape Town. South Africa. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Merced M Leiker Research Technician II Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Green Fluorescent Protein
Hello, I routinely use Invitrogen's Rabbit anti-GFP on mouse and rat FFPE tissue. Paula From: Melanie Black melanie.bl...@uct.ac.za To: histonet@lists.utsouthwestern.edu Sent: Tue, October 27, 2009 7:48:48 AM Subject: [Histonet] Green Fluorescent Protein Hi I am looking to demonstrate Green fluorescent protein in Para formaldehyde fixed, processed rat tissue. Apart from using an antibody against GFP, can the native GFP be detected? Gayle Callis, I believe you may be able to help me with this method. Thanks Melanie Melanie Black 082 469 3352 Cardiovascular Research Unit 3rd Floor; Chris Barnard Building Medical School; Observatory. 7925. University of Cape Town. South Africa. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Problem wih FITC secondary unbinding after about 10 hours (using immunofluorescence)
We are having a very strange problem with our FITC-conjugated secondary in double labeling immunofluorescence processing. We are processing for 3 different enzymes using a Rhodamine secondary and these seem to be fine throughout the process, but using 3 different markers (GFAP, MAP2, OX42) with a FITC conjugated secondary we see something very strange. The labeling looks really nice immediately after processing and mounting for up to 6-10 hours later, then when left in the fridge overnight, the next day the FITC (and only the FITC, the rhodamine is fine) seems to unbind and is all over the tissue creating a background type fluorescence. I have tried 4 different secondaries from 3 different companies (although they are all FITC conjugated - want to try a cy2 or alexafluor 488 but don't have any yet) and they all do they same thing - really nice immediately after processing, but cell labeling is gone by the next day and appears as specks all over the tissue sample. We are using 4%PFA fixed, floating rat brain sections (used three different brains so far and all had the same effect). These are the other things I checked: - used 3 different mounting media (Fluoromount-G, Elvanol, 90% glycerol): all had same effect. Leaving tissue overnight in PBS in fridge also had same effect, so figure it cant be anything with the mounting process. - used 3 different primaries and all have same effect - we use ovoid (BR0014G) PBS tablets (1 per 100ml, pH 7.3) - commercially available standard PBS tablets... should be okay?? Anyone else use this? Any help would be appreciated, or just to know if anyone has seen this before. I did immuno for 5 years in a different lab and never saw anything like this, but recently switched labs and had this problem immediately. I figure it must be something in the solutions I am using n the new lab, but I just cant fathom what it would be at this point. Thanks Dr. Janelle Pakan University of British Columbia Brain Research Centre 2211 Westbrook Mall Vancouver, BC Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Problem wih FITC secondary unbinding after about 10 hours (using immunofluorescence)
We use a range of Alexa Fluors (including 488) all the time with either SlowFade Gold or ProLong Gold antifade mounting media on every primary antibody we use. We can still see the label a week or more later, especially if we store the slides at -20 C. Alexa Fluors are very bright and stable compared to traditional fluorophores like FITC, TRITC... Another consideration is are you doing confocal or epifluorescent imaging? Confocal will bleach your sample sooner. The Alexa Fluors and mounting media mentioned above are all available from Invitrogen at the following links (this is NOT a plug for Invitrogen, though it may appear that way; we are an independent lab!): http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/alexa-fluor.html http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/SlowFade-Gold.html http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/ProLong-Antifades-Brand-Page.html Regards, Merced M Leiker Research Technician II Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. --On Tuesday, October 27, 2009 10:36 AM -0700 Janelle Pakan janelle.pa...@gmail.com wrote: We are having a very strange problem with our FITC-conjugated secondary in double labeling immunofluorescence processing. We are processing for 3 different enzymes using a Rhodamine secondary and these seem to be fine throughout the process, but using 3 different markers (GFAP, MAP2, OX42) with a FITC conjugated secondary we see something very strange. The labeling looks really nice immediately after processing and mounting for up to 6-10 hours later, then when left in the fridge overnight, the next day the FITC (and only the FITC, the rhodamine is fine) seems to unbind and is all over the tissue creating a background type fluorescence. I have tried 4 different secondaries from 3 different companies (although they are all FITC conjugated - want to try a cy2 or alexafluor 488 but don't have any yet) and they all do they same thing - really nice immediately after processing, but cell labeling is gone by the next day and appears as specks all over the tissue sample. We are using 4%PFA fixed, floating rat brain sections (used three different brains so far and all had the same effect). These are the other things I checked: - used 3 different mounting media (Fluoromount-G, Elvanol, 90% glycerol): all had same effect. Leaving tissue overnight in PBS in fridge also had same effect, so figure it cant be anything with the mounting process. - used 3 different primaries and all have same effect - we use ovoid (BR0014G) PBS tablets (1 per 100ml, pH 7.3) - commercially available standard PBS tablets... should be okay?? Anyone else use this? Any help would be appreciated, or just to know if anyone has seen this before. I did immuno for 5 years in a different lab and never saw anything like this, but recently switched labs and had this problem immediately. I figure it must be something in the solutions I am using n the new lab, but I just cant fathom what it would be at this point. Thanks Dr. Janelle Pakan University of British Columbia Brain Research Centre 2211 Westbrook Mall Vancouver, BC Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Merced M Leiker Research Technician II Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Green Fluorescent Protein
At the suggestion from someone on Histonet, I've been using Abcam's chicken anti-GFP. It's worked great on mouse tissue fixed with paraformaldehyde, Zn buffered formalin, or 10% neutral buffered formalin. Even though Abcam suggests that you antigen retrieve, I've found it works just as well without it. As for viewing GFP without any antibody, you apparently can, at least in bone. See http://skeletalbiology.uchc.edu/30_ResearchProgram/304_gap/index.htm for a really detailed discussion. I've tried before and it hasn't worked... I think you need really high quality optics and filter cubes. Adam On Tue, Oct 27, 2009 at 8:50 AM, Paula Pierce cont...@excaliburpathology.com wrote: Hello, I routinely use Invitrogen's Rabbit anti-GFP on mouse and rat FFPE tissue. Paula From: Melanie Black melanie.bl...@uct.ac.za To: histonet@lists.utsouthwestern.edu Sent: Tue, October 27, 2009 7:48:48 AM Subject: [Histonet] Green Fluorescent Protein Hi I am looking to demonstrate Green fluorescent protein in Para formaldehyde fixed, processed rat tissue. Apart from using an antibody against GFP, can the native GFP be detected? Gayle Callis, I believe you may be able to help me with this method. Thanks Melanie Melanie Black 082 469 3352 Cardiovascular Research Unit 3rd Floor; Chris Barnard Building Medical School; Observatory. 7925. University of Cape Town. South Africa. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Green Fluorescent Protein
GFP can be detected directly in FFPwax sections if the expression level is high enough. Yo can easliy check this, using one section before you immunoprobe. Sure, Ab detection will guarantee you a strong signal but, again, if the exression of the protein is high enough. I have come across several tissues where I can't even detect GFP using an Ab. I agree that both Invitrogen's ( mouse A11120 and rabbit A11122 ) anti GFP Abs are very good. So also is Abcam's chicken anti GFP ab13970 : for me it gives the best signal but, you'd specifically need to buy an anti Chicken IgY conjugate. If you care to browse in the Image gallery here: http://www.immunoportal.com/index.php, there are some images. good luck! Carl ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Quick help with fixative?
Why would lymph nodes for AFB be submitted in saturated sodium borate? I'm cutting in now and want to know why this would be! Sally Breeden, HT(ASCP) NM Dept. of Agriculture Veterinary Diagnostic Services PO Box 4700 Albuquerque, NM 87106 505-841-2576 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Problem wih FITC secondary unbinding after about 10 hours (using immunofluorescence)
Thanks Merced, We are doing confocal, but the problem doesnt seem to be bleaching - the fluorescence is still there, it is just no longer concentrated within the cells we are staining for, but rather dispersed all over the tissue. Almost like it leaked out of the cells we were staining. As I mentioned, I dont think it can be the mounting media, as when we left the tissue in PBS overnight without mounting it, the signal was still gone the next day. Must be some sort of solution problem... maybe salts, pH, fixative?? But we are using standard protocols and comercially available PBS. I am stumped... Dr. Janelle Pakan University of British Columbia Brain Research Centre 2211 Westbrook Mall Vancouver, BC Canada On Tue, Oct 27, 2009 at 11:29 AM, Merced M Leiker lei...@buffalo.eduwrote: We use a range of Alexa Fluors (including 488) all the time with either SlowFade Gold or ProLong Gold antifade mounting media on every primary antibody we use. We can still see the label a week or more later, especially if we store the slides at -20 C. Alexa Fluors are very bright and stable compared to traditional fluorophores like FITC, TRITC... Another consideration is are you doing confocal or epifluorescent imaging? Confocal will bleach your sample sooner. The Alexa Fluors and mounting media mentioned above are all available from Invitrogen at the following links (this is NOT a plug for Invitrogen, though it may appear that way; we are an independent lab!): http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/alexa-fluor.html http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/SlowFade-Gold.html http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/ProLong-Antifades-Brand-Page.html Regards, Merced M Leiker Research Technician II Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. --On Tuesday, October 27, 2009 10:36 AM -0700 Janelle Pakan janelle.pa...@gmail.com wrote: We are having a very strange problem with our FITC-conjugated secondary in double labeling immunofluorescence processing. We are processing for 3 different enzymes using a Rhodamine secondary and these seem to be fine throughout the process, but using 3 different markers (GFAP, MAP2, OX42) with a FITC conjugated secondary we see something very strange. The labeling looks really nice immediately after processing and mounting for up to 6-10 hours later, then when left in the fridge overnight, the next day the FITC (and only the FITC, the rhodamine is fine) seems to unbind and is all over the tissue creating a background type fluorescence. I have tried 4 different secondaries from 3 different companies (although they are all FITC conjugated - want to try a cy2 or alexafluor 488 but don't have any yet) and they all do they same thing - really nice immediately after processing, but cell labeling is gone by the next day and appears as specks all over the tissue sample. We are using 4%PFA fixed, floating rat brain sections (used three different brains so far and all had the same effect). These are the other things I checked: - used 3 different mounting media (Fluoromount-G, Elvanol, 90% glycerol): all had same effect. Leaving tissue overnight in PBS in fridge also had same effect, so figure it cant be anything with the mounting process. - used 3 different primaries and all have same effect - we use ovoid (BR0014G) PBS tablets (1 per 100ml, pH 7.3) - commercially available standard PBS tablets... should be okay?? Anyone else use this? Any help would be appreciated, or just to know if anyone has seen this before. I did immuno for 5 years in a different lab and never saw anything like this, but recently switched labs and had this problem immediately. I figure it must be something in the solutions I am using n the new lab, but I just cant fathom what it would be at this point. Thanks Dr. Janelle Pakan University of British Columbia Brain Research Centre 2211 Westbrook Mall Vancouver, BC Canada ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Merced M Leiker Research Technician II Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu
[Histonet] Sodium Borate question resolved
Thanks to all who responded to my sodium borate question. I've been helped and am Internally Grateful (whatever...). Sally Breeden, HT(ASCP) NM Dept. of Agriculture Veterinary Diagnostic Services PO Box 4700 Albuquerque, NM 87106 505-841-2576 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Equipment maintenance companies in Phoenix AZ?
Hi Netters,Does anyone know of a good histology equipment maintenance company in phoenix AZ? Annual maintenance is due so need to schedule soon. Thanks in advance.. Jill Cox HT (ASCP) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] HT (ASCP) Position Available
I have a full time HT (ASCP) position available at a mid-sized Medical Center in Manchester, NH. This is a hospital based start up opportunity in a brand new lab that starts Dec 1. This is a day shift position. If you are interested apply at: http://www.catholicmedicalcenter.org/Career/JobSearch.aspx or email me sfe...@cmc-nh.org Thanks, Steve Feher Stephen A. Feher, MS, SCT (ASCP) Pathology Supervisor Catholic Medical Center 100 McGregor Street Manchester, NH 03102 603-663-6707 sfe...@cmc-nh.org mailto:sfe...@cmc-nh.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Unsubscribe
Unsubscribe ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
