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By John R. Gordon
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Extra resources for A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition
Wash the plates 8 - 10 times with distilled H2O, making sure to remove excess fluid. Add 100 µl of ABTS substrate to each well and allow the reactions to develop for 20 - 45 min at room temperature. The reactions can be stopped by 7. adding 100 µl of 1% SDS to each well. Read the plates using the ELISA plate reader, set at a reading wavelength of 405 nm. 7 In vivo assessment of T cell responses: Th1 versus Th2 responses Materials Ovalbumin- and SRBC-immune mice Tissue processor (for processing biopsies to paraffin blocks) 1 ml syringes and 30 ga needles scalpel blades Reagents Ovalbumin in Ca++/Mg++-free HBSS (40 µg/ml) SRBC in Ca++/Mg++-free HBSS (5% suspension) In situ hybridization fixative 70% ethanol METHOD 1.
This procedure is used to detect the abilities of individual B or T (or other) cells to secrete their products. We will assess the abilities of splenocytes from BALB/c mice that have been immunized with ovalbumin-alum to produce ovalbumin-specific antibodies of varying isotypes or those of these mice or SRBC-vaccinated mice to produce IL-4 or IFN-γ in response to antigenic challenge in vitro. , Th1 or Th2) of the mice to these immunogens. 05% Tween 20) streptavidin-alkaline phosphatase conjugate (strep-AP; diluted 1:5000 in PBST) METHOD 1.
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. 62 8. Place the tissues into labelled tissue-tek cassettes and transfer into the tissue processor at the 70% ethanol step. The processor will automatically process the tissue through to molten paraffin. 9. 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.
A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition by John R. Gordon