Processing of tissue culture cells.
Live cells are often studied, in vitro, bathed in nutrient growth media. It is sometimes necessary to study individual compounds produced by these cells and antibodies work well for this purpose. These cells must first be concentrated and fixed before testing. There are a couple of ways to study cells in culture using antibodies. One is to fix the cells in place as they adhere to a solid surface and then test them as though they were cells on a slide. Another is to retrieve them and pellet the cells, fixing them in a test tube and then embedding and sectioning them as though they were a solid tissue. Fixatives can be mild to moderate depending on the antigens to be studied. Sectioned cells can be tested following mild pretreatment steps. Cells fixed in the culture dish can be tested following mild pretreatment steps in buffers containing 0.25% Triton X-100 and 5% dimethylsulfoxide (DMSO) to allow for easier antibody penetration. The endogenous peroxidase enzyme or oxidative compounds can be quenched in a mild hydrogen peroxide solution. The sections are then ready to test with antibody after an incubation in a normal serum solution blocks any available charged sites.
Bratthauer
Gary L
GL
Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, Washington, DC, USA.
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005404:Fixatives / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D017830:Octoxynol / Q000737:chemistry; D009195:Peroxidase / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*
D000906:Antibodies; D005404:Fixatives; D017830:Octoxynol; D006861:Hydrogen Peroxide; D009195:Peroxidase; D004121:Dimethyl Sulfoxide