By John R. Gordon
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Extra resources for A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition
2. Allow the cells to continue growing until the medium has gone completely yellow (acidic) and the cells have essentially all died (about 5 - 7 days; terminal cultures). 3. Collect the supernatant from each flask and centrifuge it for 15 min at 12,000 rpm in the RC-5B superspeed centrifuge to sediment all particulate matter. 4. Aliquot the supernatants and either process expeditiously or store at -20˙C or 80˙C (for longer-term storage). 1 Avid-AL affinity chromatography AVID-AL is a popular new matrix with a natural affinity for IgG from a wide array of species, including rat.
8. 8 and then pour off the alcohol overlay and rinse the top of the gel with water. 5 ml of the stacking gel buffer, and degas the solution under vacuum for 10 - 15 min. Add 25 µl of ammonium persulfate, and mix once again by swirling gently, and pipette this solution on top of the polymerized separation gel. Immediately place the well-forming comb in place, with the teeth submersed in the stacking gel solution. Allow the stacking gel to polymerize, and then remove the comb from the top of the stacking gel.
Embed the tissues in paraffin such that the ear tissues are standing straight up in the molds, so that upon sectioning you will obtain cross-sections of the tissue. 10. Cut 6 µm paraffin sections of the tissues and dry them onto slides overnight at 42˙C. 11. 2), mount with permount and examine the tissues under the microscope. 5-positive CD8 cells, cytokine-secreting cells, etc). It is thus similar to FACS analysis, but has perhaps traditionally been used most often with fixed tissues or cells, allowing one to assess the prevalence of marker expression in situ, within the context of ongoing physiological or pathological processes.
A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition by John R. Gordon