Mitogen-Activated Protein Kinase: MATERIALS AND METHODS(2)


Immunofluorescence Microscopy

Cumulus-free oocytes were fixed in ice-cold 100% ethanol for 20 min, then permeabilized in PBS containing 0.1% Triton X-100 for 15 min at 38°C and washed in 0.1% Tween 20 in PBS (PBS-T). After blocking with 5% BSA in PBS (PBS-BSA) for 1 h at 38°C, the oocytes were incubated with anti-MAPK polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:20 in PBS-BSA for 2 h at 38°C, washed in PBS-T, and incubated with TRITC (tetramethylrhodamine В isothiocyanate)-conjugat-ed goat anti-rat IgG (Organon Teknika Corp., Durham, NC) diluted 1:40 in PBS-BSA for 2 h at 38°C. Chromosomes were stained in 10 mg/ml Hoechst-33342 in PBS for 20 min at 38°C. Stained oocytes were mounted on glass slides with mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and examined with a fluorescence microscope (BHS-RFC; Olympus, Tokyo, Japan). Photographs were recorded on Fujichrome Provia film (1600 ASA; Fuji Photo Film, Tokyo, Japan). ventolin inhalers

Separation of GVs

Oocytes not cultured or cultured for 25 h were denuded from cumulus cells; then, to remove their zona pellucida, they were immersed in acid Tyrode’s solution (pH 2.5) for a few seconds. After washing in mKRB containing BSA, zona-free oocytes were disrupted by gentle pipetting in lysis buffer composed of 10 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KC1, 0.2 mM PMSF, 0.5 mM dithiothreitol (DTT), 2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mg/ml pep-statin, 0.5% Triton X-100, and 0.04% trypan blue. Intact GVs were selected and washed in ice-cold extract buffer (EB; composed of 15 mM EGTA [pH 7.2], 1% Nonidet P-40, 60 mM sodium ^-glycerophosphate, 30 mM p-nitro-phenylphosphate, 25 mM 3-[7V-morpholino]propanesulfonic acid, 15 mM MgCl2, 0.2 mM Na3V04, 1 mM DTT, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mg/ml pepstatin, 1 mM PMSF, and 50 mM p-aminobenzoic acid); collected GVs were then stocked in EB at -80°C until used.

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