re: [Histonet] Immunofluorescence on FFPE skin
To me, the critical data is: which Abs do you want to use on FFPWS of skin, please? If you do chromogen IHC on your skins already, with them Abs, you can do IF. Sure, autofluorescence can be a problem but..not that big a problem. Interestedly, Carl NB: that article is..strange. Carl Hobbs FIBMS Histology and Imaging Manager Wolfson CARD School of Biomedical Sciences Kings College London Guys Campus SE1 1UL Tel: 020 78486813 Fax: 020 78486816 020 78486813 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Immunofluorescence in the clinical laboratory, some questions
Hi Gayle, We perform IF on renal biopsies and dermatological specimens with various bullous disorders. We perform IgA, IgM, IgG, C3, C1q, Albumin, Fibrinogen, Kappa and Lambda on acetone fixed frozen sections. These are all direct IF. We perform C4D indirectly on frozen tissue due to the lack of staining intensity of any directly conjugated antibodies, as well as C4D IF on FFPE tissue and we also perform IHC C4D on FFPE. Our pathologist's usual comments about the reason that they like the immunofluorescence is that they can easily see smaller deposits using immunofluorescence that would be indeterminate with conventional IHC. We use an automated stainer for these, it allows us to be more hands off as well as to incubate them in the dark. Let me know if you have any further questions. On Sat, May 12, 2012 at 7:48 AM, gayle callis gayle.cal...@bresnan.netwrote: Dear Histonetters, I know that immunofluorescence has been done for decades on renal biopsies, but am curious if laboratories are using IF more these days? If so, I would be very interested to talk to you one on one about this as I have more questions on why you deviate from standard chromogenic enzyme immunohistochemistry and perform IF. Comments about renal biopsy procedures are welcome too. Also, do you do mostly single IF or double IF, and the reasons why? Is your IF done primarily on FFPE or frozen sections/acetone fixation? When you do immunofluorescence in your clinical laboratory are you using an automated stainer or a manual protocol? Or is your clinical laboratory associated with medical research groups? Any comments/information is most welcome. Thanks.. Gayle M. Callis HTL/HT/MT(ASCP) Bozeman MT ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Patrick Laurie HT(ASCP)QIHC CellNetix Pathology Laboratories 1124 Columbia Street, Suite 200 Seattle, WA 98104 plau...@cellnetix.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] immunofluorescence mounting medium?
http://www.vectorlabs.com/catalog.aspx?catID=279 VECTASHIELD Mounting Media for Fluorescence This one works for me and was recommended by Leica technical support for oil immersion image. Original Message From: Collette, Nicole M. Sent: Mon, Mar 5, 2012 12:35 PM To: histonet@lists.utsouthwestern.edu CC: Subject: [Histonet] immunofluorescence mounting medium? Hello, Esteemed Histonetters, I am trying to get nice publication-quality images of my immunofluorescent tissue sections. I am currently using Prolong Gold, and after I let the stuff cure for several days, my 100X oil-immersion images are still smeary, even after taking into account the unevenness of the tissue section. I don't seem to have this problem with colorimetric stains (histological stains, LacZ, etc.), so I am thinking the mounting medium is part of the problem? It seems to me that it never fully cures at the middle of the coverslip… Does anyone have a recommendation for a mounting medium that works better for this purpose? Any advice is appreciated. Thanks in advance, and Happy Monday! Sincerely, Nicole Collette Lawrence Livermore National Laboratory collet...@llnl.gov ___ 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] Immunofluorescence
Paraffin does indeed give a lot of autofluorescence. We use a solution of 0.3% Sudan Black in 70% Ethanol for 10 minutes on the tissues to help with that after the staining. It doesn't mask the autofluorescence completely but reduces it enough to give us good contrast of signal against the background. Mostly we look at cardiac tissue. Maybe someone will have a better idea for brain... Regards, Merced -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Natalia Fernandez Sent: Tuesday, February 07, 2012 10:35 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Immunofluorescence Hi everybody! I have a problem with my experiment. I try to do immunofuorescences in old human brains (parafine sections). First I thought that the too much colocalisation was due to my antibodies (primary or secondary). After several tests (Only 1st antibody, only 2nd one, simple staining, inverse simple staining,..) I saw that the tissue itself shows a lot of fluorescence (without antibodies, and even after a treatment against lipofuscine (solution of Potassium permanganate). So my question is: Do you have the same problem? Do you think that parafine could be the reason of this autofluorescence?! What can I do? Thank you so much for your answers :) Natalia ___ 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] Immunofluorescence
Hi Natalia, Human tissue is naturally autofluorescent. In our lab we do immunos on frozens and quench the AF with chicago blue solution (1g Chicago Sky Blue 6B-Sigma in 99ml MilliQ plus 1ml DMSO) after washing the secondary antibodies with PBS (3x). Just load the tissue with a drop of the CB solution for 15 secs and wash it in PBS again (3x). Mount as usual D -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Natalia Fernandez Sent: Wednesday, 8 February 2012 2:35 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Immunofluorescence Hi everybody! I have a problem with my experiment. I try to do immunofuorescences in old human brains (parafine sections). First I thought that the too much colocalisation was due to my antibodies (primary or secondary). After several tests (Only 1st antibody, only 2nd one, simple staining, inverse simple staining,..) I saw that the tissue itself shows a lot of fluorescence (without antibodies, and even after a treatment against lipofuscine (solution of Potassium permanganate). So my question is: Do you have the same problem? Do you think that parafine could be the reason of this autofluorescence?! What can I do? Thank you so much for your answers :) Natalia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet __ This email has been scanned by the Symantec Email Security.cloud service. For more information please visit http://www.symanteccloud.com If you have any question, please contact MCRI IT Helpdesk for further assistance. __ __ This email has been scanned by the Symantec Email Security.cloud service. For more information please visit http://www.symanteccloud.com __ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Immunofluorescence staining/minimizing background staining
Hi, What fluorochromes are you using? There is a lot of autofluorescence in the FITC channel. Have you looked at an unstained tissues under the scope with each filter that you need, that may give you a clue as to where your background is coming from. In addition to an unstained slide, I suggest eliminating the primary to see if your secondary is sticking to the tissues. Are you doing single-color or double stains? If you are getting a lot of autofluorescence with one fluorochrome, I would suggest trying a different one. We use alexafluur-647 (far red) a lot because is it fairly clean (ie - very little autofluorescence in that channel) Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: Kasai, Miki (NIH/NCI) [E] kas...@mail.nih.gov To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Sent: Monday, January 30, 2012 4:50 PM Subject: [Histonet] Immunofluorescence staining/minimizing background staining Hi, We are performing some immunofluorescence staining on mouse lung tissue. We are getting some nice positive staining with some of our initial antibodies (procollagen, cytokeratin). We would like to minimize the amount of background staining we are getting. We are titering our primary antibodies to find out optimal Ab concentration as well as the secondary conjugate Ab with the fluorophore of interest. We use donkey serum for general blocking. Any other suggestions? Much appreciation, Miki ___ 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] Immunofluorescence double labeling with two rabbitantibodies
Aline, Jackson Immunoresearch provides protocol and reagents that are useful for solving your problem: http://www.jacksonimmuno.com/technical/fab-blok.asp I have used this approach to localize at the same sample simultaneously two different isoforms of a transcription factor with two different mouse monoclonals. Worked for me. Anatoli Gleiberman, PhD Director of Histopathology Cleveland Biolabs, Inc 73 High Street Buffalo, NY 14203 phone:716-849-6810 ext.354 fax:716-849-6817 e-mail: agleiber...@cbiolabs.com -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Aline Rodrigues Sent: Tuesday, October 12, 2010 1:29 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Immunofluorescence double labeling with two rabbitantibodies Hello all, I using formalin fixed paraffin embedded specimens from fetuses from sheep to examine which cells are virally infected within the CNS. I am using IBA-1 to identify microglial cells. The IBA-1 antibody is a rabbit antibody and it works really well in my specimens. At the same time I am also using an antibody against the virus that I am studying that is also a rabbit Ab. For these samples I am using double labeling to determine if cells are infected or not. Both antibodies work, but I can use them together otherwise there is x- reaction. Does anyone has any suggestions of how to label tissues with two different antibodies that come from the same species. On immunoportal someone suggested to use 4%PFA to fix the tissues in between the first (primary and secondary Ab) and the second (primary and secondary Ab), however it did not work. PFA did not block the previously bound Abs. The other option that I would have would be to use IBA-1 from goat. My problem with this antibody coming from goat is that I can have x- reaction and moderate background since goat and sheep are very closely related. Please, if someone has any suggestions I would immensely appreciate! Thank you very much! I hope you have a wonderful day! Aline ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This communication may contain privileged information. It is intended solely for the use of the addressee. If you are not the intended recipient, you are strictly prohibited from disclosing, copying, distributing or using any of this information. If you received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. This communication may contain nonpublic information about individuals and businesses subject to the restrictions of the Gramm-Leach-Bliley Act. You may not directly or indirectly reuse or redisclose such information for any purpose other than to provide the services for which you are receiving the information. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
