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Tissues were dissected, post-fixed for 3 h in 4% PFA at room temperature 1820 2 C cryoprotected in 30% (w/v) sucrose in Tris-Azide buffer (40 mM Tris, 10 mM phosphate, 0.7% (w/v) NaCl, 0.05% (w/v) NaN 3 ) at 4 C, then embedded in optimal cutting temperature (OCT) compound, rapidly frozen in isopentane on dry ice, and stored at 80 C until use

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In practice, many researchers use the 28-day mark as a hard cutoff

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It works over a wide pH range and is used between 0.5-1.2%