Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

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"Human lung samples or fibroblasts cultured on decellularized ECM and were formalin-fixed and paraffin-embedded (FFPE). De-paraffinized and rehydrated 4 μm sections were subjected to antigen-heat retrieval (BioCare RV1000) for 25 minutes at 100oC (then allowed to cool to room temperature for 20 minutes), followed by 10 minutes in 3% hydrogen peroxide and 1 hour in Background SNIPER (BioCare BS966) blocking reagent at room temperature. Fibroblasts cultured on 2-D hydrogels were formalin-fixed and subjected to antigen-heat retrieval followed by a Background SNIPER blocking reagent. For anti-procollagen I, we used proteinase K (Millipore 21627; working strength) for 10 minutes instead of antigen-heat retrieval. After
blocking, sections or gels were exposed overnight (4oC) to primary antibodies (Table E3) diluted in 10% Background SNIPER. For permanent staining, Novolink Polymer Detection Systems (Leica RE7270-RE; manufacturer’s recommendation) was used and developed with DAB chromagen (Covance SIG-31042; manufacturer’s recommendation). For procollagen type I, biotinylated anti-Rat (Vector Laboratories #BA-4001) was used at 1:500 in 10% Background Sniper followed by Streptavidin-HRP (Covance SIG-32254; working strength) and DAB chromagen. Slides were counterstained with hematoxylin and cover-slipped with Permount (FisherSci #SP15). For immunofluorescence, after overnight primary antibody incubation, anti-Rabbit Cy3 or anti-rat Alexa 594 (Jackson 711-165-152; Abcam ab150156) in 10% Background SNIPER was incubated for 2 hours at room temperature and cover-slipped with Prolong Gold Antifade Mountant w/ DAPI (ThermoFisher P36931). "

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