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By John R. Gordon

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6. 7. 8. , 1 ml/tube). 0). a. Bio-Rad, Bradford or CBB] protein assay; Appendix D), and pool the protein-containing fractions. Regenerate the column with ≈10 ml of PBS, and store the matrix in PBS/20% ethanol. ). After dialysis, determine the protein concentration of the eluted IgG solution using a and, if necessary, concentrate the eluted protein using a centrifugal concentrator. , protein A, T gel, Avid-Chrom), so alternate methods must be employed to prepare IgM antibodies. A number of methods are available, with the simplest true purification probably being achieved by size exclusion chromatography (IgM pentamers have a molecular mass of ≈750 kD, while IgG and albumin have molecular masses of 150 & 65 kD, respectively).

6. 7. 8. The next morning, remove the capture antibody from the wells by inverting the plate and sharply flicking it. ). Remove the blocking solution, rinse as above and add 100 µl of fresh blocking solution to each well. Incubate the plates at 37°C for a minimum of 1 hour (or until the cells from the next step are ready). Generate a single cell suspension from the spleens of an OVA- and SRBCsensitized mice, and resuspend the cells to 1x107 cells/ml DMEM-10% FCS. Remove the ELISPOT plate from the incubator and dump out the blocking solution.

5. , one SRBC- and one ovalbumin-sensitized mouse) and take biopsies of the reaction sites. To do this, use a fresh #12 scalpel blade to remove the ear and cut it in half longitudinally, through the middle of the reaction (injection) site. Trim away and discard the tissue from the outside of the ear, away from the injection sites. 6. Transfer each half of the ear into ice-cold ISH fixative and fix the tissue for 3 h on ice. 7. Replace the ISH fixative with ice-cold 70% ethanol and store the tissue in this solution at -20˙C until you are ready for tissue processing.

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