Hello flowers Two questions about cytoplasmic staining I wanted to confirm that when cells are fixed with formaldehyde, are all cytoplasmic proteins are fixed as well? And would therefore any antibody used to stain cytoplasmic proteins have to be able to recognize fixed proteins and a "fixed" epitope? Is that a big problem and could it be avoided by fixing first, then permeabilizing without any fixative in the solution? And, part two, I tried using the control of pre-incubating a polyclonal antibody with the recombinant chemokine and then stained the cells with the mixture, but what I got was actually higher fluorescence than in the cells stained with the antibody alone. Does this mean the conjugates got stuck inside of the cells? Any help would be greatly appreciated ********************** Artur Plett Post-doctoral fellow IU Sch. of Medicine Div. of Hematology/Oncology 1044 W. Walnut St Indianapolis, IN 46202 phone: 317-274-0352 fax: 317-278-3538 -----Original Message----- From: Calman Prussin [mailto:CPRUSSIN@niaid.nih.gov] Sent: Thursday, September 13, 2001 9:49 AM To: cyto-inbox Subject: RE: More about cytoplasmic staining. This is not a control too far! A substantial fraction of the intracellular cytokine staining that I see appears to be artifact. Blocked controls such as you describe are one of the best ways to verify your data and avoid such artifacts. Intracellular staining with a new mAb or new system should be validated 2-3 times using either an unlabelled mAb or recombinant cytokine blocked control. Isotype controls by themselves are not adequate and should only be used once you have performed more rigorous blocking controls. Different mAbs posses different non-specific binding characteristics, thus some isotype matched controls may poorly match the non-specific bonding of you anti-cytokine mAb. Fixed permeabilized cells demonstrate far greater non-specific binding than fresh cells and thus require more stringent controls. I have typically used 100 mcg/ml of unlabeled mAb (staining in 50 ul) to block and then add the labeled anti-cytokine mAb to the blocked cells WITHOUT washing out the unlabeled mAb. The correct positive control for this is to block with 100 mcg of isotype control, then stain with the labeled anti-cytokine mAb. The most rigorous control is to stain with 2 different mAbs that recognize 2 different mAbs on the intracellular Ag, demonstrate they recognize the same population and then block each separately (Prussin, J Immunol Methods 188: 117-128) A more detailed discussion of these issues is presented in Unit 6.24 of current Protocols in Immunology. Calman _______________________ Calman Prussin Laboratory of Allergic Diseases NIAID/ National Institutes of Health Building 10, Room 11C205, MSC-1881 Bethesda, MD 20892-1881 (301) 496-1306 (Voice) (301) 496-1306 (Fax) calman@nih.gov ---------- From: Finney, Simon Sent: Wednesday, September 12, 2001 5:52 To: cyto-inbox Subject: More about cytoplasmic staining. Dear all, There has been a bit of discussion about intracellular staining recently with primary conjugated antibodies. I was trying to prove specificity of my intracellular binding by blocking with unconjugated antibody first. My conjugated antibody still appears to bind but I am unclear as to appropriate doses to block with and whether the conjugated one can swap places with unconjugated one during the second incubation. Is this a control too far ? Isotype controls (admittedly from a different company) show no binding. Thanks Dr Simon Finney Unit of Critical Care NHLI at Imperial College London UK
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