Rat Amnion Type IV Collagen Composition and Metabolism: MATERIALS AND METHODS(1)

2 May
2013

MATERIALS AND METHODS(1)

Animals

Timed pregnant Sprague-Dawley rats (Zivic-Miller, Port-ersville, PA) were killed on Days 18-21 of pregnancy. The animals usually deliver on the afternoon of Day 21. Amnions were collected as previously described. Tissue extracts prepared in a buffer consisting of 50 mM Tris, 1% Nonidet NP-40, 0.1% deoxycholate, 0.1% SDS, 150 mM NaCl, 10 ^g/ml aprotinin, and 1 mM EDTA were used for Western blot analysis as previously described. Extracts made in 50 mM Tris buffer containing 100 mM CaCl2 and 150 mM NaCl, pH 7.4, were used for zymography. All experiments were performed on at least two separate occasions.

Antibodies

Polyclonal antibodies raised against the C-terminal non-collagenous globular domains (NC1 domains) of specific alpha chains have been described previously. Polyclonal antibodies against MMP-2 (Ab17) and MMP-9 were generously provided by William Stetler-Steven-son (National Institutes of Health) and Rafi Fridman (Wayne State University, Detroit, MI), respectively. buy yasmin online

Western Blotting

Western blot analyses were carried out as previously reported. Briefly, tissue extracts (50-80 ^g protein/ lane) were separated by SDS PAGE, and the separated proteins were then transferred to membranes and probed with rabbit polyclonal antibodies. Antigen-antibody complexes bound to the membranes were detected using the enhanced chemiluminescence (ECL) reagent system (Amersham, Pis-cataway, NJ).

top