BrdU检测protocol-Bromodeoxyuridine Immunohistochemistry.doc

Bromodeoxyuridine Immunohistochemistry Sharon Morgenbesser, 10/15/93Revised by Nanette J. Liégeois 7/1/96 Introduction: This method for the detection of cellular proliferation includes several modifications of a previously published protocol (Hayashi, et al., 1988, J. Histochem. Cytochem. 36:511-514). This method utilizes formalin-fixation, paraffin-embedding, and an anti-BrDU mAb that detects BrDU in ssDNA after denaturation and neutralization. Because it has been optimized for midgestational murine lens analysis, it may require further adjustment for other tissues. Abbreviations: e (embryonic day), RT (room temp.), EtOH (ethanol), O/N (overnight) Method: A. BrDU Treatment, Fixation, and Sectioning: 1. Prepare Bromodeoxyuridine: 10mg/ml in 1X PBS. Make up BrDU in a light-protected conical and solubilize at 37°C. Estimate the amount of BrDU at prepare by assuming that each mouse is 40g. 2. Inject BrDU into a pregnant mouse intraperitoneally at 100 μg BrDU/g body weight (Miller and Nowakowski, 1988, Brain Res. 457: 44-52) using a 26 gauge needle and a 1cc syringe. 3. Embryos are removed from the mother and placed quickly in a dish of ice-cold 1XPBS. 4. Fix embryo heads for 2 hr RT. Tissue samples are placed in a scintillation vial containing apx. 20ml 10% buffered formalin. For e14.5 and older embryos, the head should be removed and fixed separately from the body. Place on an orbital shaker such that the sample moves slightly. The sample must settle to the bottom. 5. Rinse with 1XPBS Gently pour off the fixative into biohazard container. Do not let the samples dry between the following steps, and use apx. 20 ml for each reagent (step 3-7). 6. 30%, 50%, 70%, 95%, 100% x 2, RT, 30 min. each, with slight agitation. This step functions to dehydrate the tissues through an increasing ethanol series: Samples in 70%, 95%, or 100% may be stored O/N, 4°C if time does not permit completion through at least one paraffin embedding step (step 6). 7. EtOH/xylene (1:1), RT; xylene