Role of nitric oxide synthase types II and III in early protection against endotoxin-induced lung injury: ANIMALS AND METHODS (part 5)

ANIMALS AND METHODS (part 5)

In situ hybridization histochemistry: Formalin-fixed, paraffin wax-embedded lung tissue was prepared as described previously . In situ hybridization was performed according to the methods described by Nouvo . Briefly, lung tissues fixed for 16 h in 10% formalin were embedded in paraffin, and 3 to 5 |im sections were put on silane-coated glass slides (Sigma). Paraffin was removed with xylene followed by serial ethanol washes, and the slide was air dried. Slides were then immersed in phosphate-buffered saline (PBS) containing proteinase K (1 |ig/mL) for 15 mins, and proteases were inactivated by washing with 0.1 M Tris (pH=7.5) and 0.1 M NaCl for 3 to 5 mins. Then, the following ingredients were put into an Eppendorf tube: 50 |iL deionized formamide (Sigma), 30 |iL 25% dextran sulphate (Sigma), 10 |iL 20x sodium chloride/sodium citrate buffer and 10 |iL DIG-labelled cDNA probe (5 to 10 |ig) obtained as described above. After vortex-ing the probe cocktail, 5 to 10 ^L were overlaid to tissue section. Slides were placed on a hot plate (95 to 100°C) for 2 to 5 mins then in a humidity chamber at 42°C overnight under coverslips. After removing the coverslips, posthybridization washes were followed by 1 h blocking with PBS and 1% albumin (Sigma). For immunological detection, slides were incubated for 2 h with a 1 to 500 dilution of polyclonal anti-DIG Fab fragments conjugated to alkaline phosphatase (Boehrin-ger Mannheim). After washing in 0.1 M Tris (pH 7.5) containing 0.1 M NaCl, the slides were incubated at room temperature in a solution of nitroblue tetrazolium and x-pho-sphate solution (Boehringer Mannheim) . Incubated slides were periodically checked under a microscope. The colour reaction was stopped by additional washing with Tris buffer containing 1 mM EDTA, and slides were coverslipped with permount. buy asthma inhalers

This entry was posted in Lung injury and tagged 1-Amino-2-hydroxy-guanidine, Endotoxic shock, NG-nitro-L-arginine, Nitric oxide, Nitric oxide synthase type II, Nitric oxide synthase type III, S-nitroso-N-acetyl-penicillamine.