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Add 100 µl of 1% paraformaldehyde and fix the cells on ice for 30 min, then wash the cells one time with PBS and resuspend to 100 µl with PBS. Store the cells in the refrigerator overnight, for FACS analysis the next day. 1 to purify all of the CD4+ or CD8+ cells, respectively, from a single cell suspension of mouse splenocytes. The real advantage of this system is that the selected populations are not killed or otherwise damaged by the isolation procedure, so that it may be possible to use them for functional studies after the purification procedure -- this is what we do in our lab to purify mast cells from tissues.

Two ovalbumin-sensitized mice and two SRBC-sensitized mice), so that it can be injected intradermally in the ear. If this will take some time, also set up a 15 ml tube (with cotton batting and methoxyfluorane) that you can use during the procedure, in order to keep the mouse anaesthetized on the bench. 3. Inject 25 µl of the 5% SRBC suspension intradermally into the right ear of the mouse and inject 25 µl of the 40 µg/ml ovalbumin solution intradermally into the left ear. 4. Return the mouse to its cage, making sure that you keep it warm (use a heat lamp if necessary) and safe (from any overly dominant littermates) during recovery.

Derivatized nylon), to which they may differentially bind, and then the protein bands on the membranes are visualized immunochemically, using alkaline phosphatase- or horse radish peroxidase-labelled antibodies and appropriate chromogens. Western blotting is a very sensitive method of detecting proteins to which antibodies already exist. 05% Tween 20) bromo-chloryl-indoyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate streptavidin-alkaline phosphatase conjugate (strep-AP; diluted 1:5000 in PBST) METHOD 1.

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A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition by John R. Gordon

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