Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
polyclonal antibody to human AAT. (The Binding Site) and visualized with AP-Red (Zymed, 
San Francisco, CA). 
In situ hybridization Wc will use an iji situ hybridization technique similar to that 
published by Prosser et al. (27), Quagliano et al. (28) and Pelton et al. (29). Wherever possible 
lungs will be perfused free of blood and the inflated during fixation. Immediately on removal, 
tissue or cells are placed in f reshly made 4% paraformaldehyde in PBS at 4°C for 12-24 hours. 
Tissue is then dehydrated through graded ethanol and xylene and embedded in paraffin wax. 
Five /im thick sections are cut, floated on DEPC distilled water, mounted on acid-washed, poly 
L-lysine coated glass slides and dried for 2 hours at 55°C. Sections are then deparaffinized in 
xylene, rehydrated through graded alcohols and left in PBS for 5 min. Sections are treated with 
proteinase K for 125 min at 37"C followed by treatment with O.IM triethanolamine buffer, pH 
8.0 for 5 min and then freshly prepared triethanolamine buffer containing 0.25% acetic 
anhydride. Slides are then washed twice in 2xSCC, dehydrated through alcohols and air dried. 
We plan to use sense and antisense riboprobes (approximately 200-300 bases in length) 
generated from the desired cDNA. The sections will be pre hybridized at 42°C for 1 h and then 
hybridized with ^“'S-labeled probe, incubated overnight in a humidified chamber at 42°C. After 
incubation, slides will be washed, dehydrated through alcohols, air dried, dipped in Kodak 
NTB-2 emulsion and stored at 4°C for 1-2 weeks. Slides will then be developed and stained and 
viewed with both bright and dark field illumination. Controls for the ^^S-labeled antisense 
riboprobes will include unrelated oligomers and sense riboprobes as appropriate. 
Northern analyses Total cellular RN A will be isolated f rom tissues by standard methods 
(30). To detect RNA from the transfected gene, total RNA will be size fractionated in a 
formaldehyde/agarose gel and the blot transferred to a nitrocellulose membrane. The blot will 
be hybridized with a probe specific for human AAT, autoradiograms will be performed and 
quantitated by laser scanner. 
Western analyses Total protein concentration in lavage fluid or tissue extracts will be 
determined by the Coomasie Blue method of Read and Northcote (31). Aliquots of the extracts 
will be subjected to polyacrylamide gel electrophoresis (PAGE) under denaturing conditions 
using standard methods (32). Following electrophoresis, the protein will be transferred 
electrically to a sheet of nitrocellulose in a Transblot chamber (Bio-Rad, Inc.) and 
immunodetection performed by published methods (33). Appropriate first and second antisera 
will be obtained from The Binding Site, San Diego, CA. Wc will use a second antiserum with 
an alkaline phosphatase conjugated group. The substrate cleaved by the alkaline phosphatase 
is 5-bromo-4-indoyl phosphate which yields a chromogcnic product in the presence of nitroblue 
tetrazolium. 
[188] 
Recombinant DNA Research, Volume 19 
