From lefortb <@t> crhsc.umontreal.ca Sun Sep 29 09:01:49 2002 From: lefortb <@t> crhsc.umontreal.ca (Bertrand Lefort) Date: Fri Sep 16 15:21:57 2005 Subject: [Histonet] Immunocytofluorescence on smooth muscle cells Message-ID: Hello, We are a new lab and we try to develop Immuno-cyto-fluorescence techniques in the lab. We are working with human bronchial smooth muscle cells. I have currently a very big problem with all rabbit antibodies. All rabbit antibodies (including IgG as isotype) give a non-specific signal, signal in the nucleus and cytoplasm with very high intensity. There is no signal between cells. This problem does not exist with Rat and mouse antibodies I have used. - I have tried different fixation methods (PFA 4%, acetone-methanol (1/1), and kit like permeafix). - I have tried different blocking solution (Rabbit serum 2%, FBS 2%, Horse serum and Universal blocker solution from Dako) without any results. - I have tried different diluents for my antibody (PBS 1X, PBS 1x-BSA 3%, Dako diluents) - I have tried different permeabilization methods (saponin, ptwx) (Not with acetone-methanol fixation). All I have tried gives always the same signal with a very high intensity signal. More details : I use a biotin-Goat-anti-rabbit for secondary antibody that give not a signal if first antibody is omitted. I use PBS to dilute my secondary antibody. I use Streptavidin-alexa to detect the signal. I have also worked on endogen biotin on this cells and it does not appear to be the problem. Has someone reported this kind of problems on human smooth muscle cells ? Has someone suggestions or solutions to this problem ? Thanks in advance Bertrand Bertrand Lefort Research Assistant Laboratory of Neuro-immunology of Asthma Research Center, Room J3155 5400, Blv. Gouin West Montreal, QC H4J 1C5 From lefortb <@t> crhsc.umontreal.ca Sun Sep 29 09:39:28 2002 From: lefortb <@t> crhsc.umontreal.ca (Bertrand Lefort) Date: Fri Sep 16 15:21:57 2005 Subject: [Histonet] Notes about my last message on smooth muscle cells Message-ID: Object : notes about message : Immunocytofluorescence on smooth muscle cells. I have few answers and it seems that my message is a little bit confused. We are studing different receptors and their effectors on smooth muscle cells. Most of antibodies we have are rabbit antibodies. Thanks in advance and sorry for the confused mail. Bertrand Bertrand Lefort Research Assistant Laboratory of Neuro-immunology of Asthma Research Center, Room J3155 5400, Blv. Gouin West Montreal, QC H4J 1C5