[Histonet] CD4 T cells FFPE vs frozen
Hi, I am aware that there are mouse anti human CD4 markers for the detection of CD4 T cells in human FFPE tissue. My question is do these antibodies (mouse clone 1F6 and 4B12) detect the same number of T cells that are detected in frozen tissue? Secondly does anyone know of any literature that validates or tests this? This question arises out of the discrepany I have noticed in our area of research(glomerulonephritis) in literature on the reported number of T cells- papers using frozen tissue report 3 times the amount of cells in the same disease studied on FFPE tissue. Any help or any anectodal evidence would be appreciated. Kim O'Sullivan -- Kim O'Sullivan Centre for Inflammatory Diseases Department of Medicine Southern Clinical School Monash Medical Centre Level 5, Block E 246 Clayton Road Clayton, Melbourne VIC 3168 Australia Ph:+61395945549 Fax:+6195946495 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] non specific staining
Hi, I am having a problem using DAB black on snap frozen mouse kidneys. We routinely stain for leukocytes (CD4(Gk1.5),neutrophils(GR1), and macrophages (fA11)) on PLP fixed tissue with very little background staining. However we currrently have to stain some snap tissue as the PLP tissue is all gone. The problem we are having is some cells are staining very specifically on the isotype controls, and controls with no primary or secondary antibody-this population of cells are hard to distinguish from positive cells in the experimental tissue. I presume it is the DAB chromagen that is binding very specifically to a certain cell population. We currently use 0.3% H202 in methanol for 30 minutes to block endogenous peroxidase- is this insufficient? Does anyone think it is an endogenous peroxidase problem or something else? Any suggestions would be helpful, Kim Department Of Medicine Monash University Monash Medical Centre Melbourne, Australia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] shrinking glomeruli
Hi, Does anyone have any suggestions as to why our mouse glomeruli are shrinking after coverlipping in DPX? Immunohistochemistry is performed on PLP frozen cryostat sections, and the glomeruli and tubules look fine upon checking after the chromagen is developed, but once they are dehydrated, cleared in histosol, and then coverslipped in DPX(also tried with Vector hardmount) the glomeruli shrink, and the tubules look like they are spreading out. We have been doing this for years, and not had a problem. Does anyone know why we would have one now? Could the disease state of the tissue contribute? Or earlier fixation- could that contribute downstream? Any suggestions would be appreciated- it is doing my head in! Kim O'Sullivan Department of Medicine Monash Univeristy Australia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Mast cell staining HELP!
Hi, Can anyone out there recommend the best way to stain mouse mast cells in the kidney(toluidine blue/or berberine sulfate) with a protocol that produces cconsistent results? Secondly, I have mouse kidney sections fixed in FFPE, PLP and methyl Carnoys. Does anyone know of a company that supplies antibodies which will work on any of these fixatives,for the staining of the IgE receptor, mast cell trytptase and chymase. Any advice would be appreciated! Kim O'Sullivan Monash University Melbourne Australia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Natural Killer Cell antibody
Hi, Does anyone out there know if there is a monoclonal anti human NK cell antibody that is exclusive, ie does not also mark CD8 T cells etc? Kim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] double immunoenzymatic methods
Hi, Have been having trouble getting an antibody to work for double immunofluoresecence (with the confocal microscope), but am able to get it to work on FFPE tissue with DAB black. Does anyone out there have a method I can use that will show colocalisation using enzyme labelling methods, ie which colour choices do you make to show double labelling within the same structure I understand this is not the preferred method. Regards Kim O'Sullivan ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
