Calcium-Dependent Actin Filament: MATERIALS AND METHODS(4)

Preparation of Tissues for Immunolocalization of Scinderin

Testes obtained immediately after death were fixed by perfusion through the testicular artery of 15 ml of PBS pH 7.4, to flush the blood out, followed by 60 ml of Bouin’s fixative. Washing the tissue with PBS was indispensable because the acetic acid contained in the Bouin’s fixative caused the blood to clot on contact, therefore blocking the vasculature of the testis and preventing the aldehyde mixture to reach the tissue. Perfusion-fixed testicular tissues were further immersed in the same fixative mixture for an additional 36-48 h at RT. For the adrenal glands, the aorta, and the vena cava, tissue fragments were immersion-fixed in Bouin’s solution. Tissues were dehydrated in ethanol and cleared in xylene before paraffinization. Five-micrometer-thick sections were mounted on glass slides coated with 3-aminopropyltriethoxysilane (Sigma), depar-affinized, and rehydrated in xylene and ethanol.

To inhibit potential endogenous peroxidase activity, tissue sections were exposed to 0.6% hydrogen peroxide (H2O2) in 70% ethanol for 5 min. They were then washed for 5 min in TBS containing 0.1% Tween-20 (TBST). Inactivation of residual picric acid was achieved using a solution of 1% lithium carbonate in 70% ethanol, and free aldehydes were blocked with a 300 mM glycine aqueous solution (pH 7.4). Spermatozoa were spotted on coated slides, air-dried, and either fixed for 5 min with 3.7% formaldehyde, washed and exposed 5 min to cold (-20°C) acetone, or treated with cold (-20°C) methanol for 5 min followed by cold (-20°C) acetone for 2 min. An exposure of the spermatozoa to methanol followed by acetone gave the best and most consistent results.

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