Lung tissues sections were prepared as described previously (9 (link), 20 (link)). In addition, endogenous peroxidase activity was quenched with 3%(v/v) H202 in methanol for 20 min and then blocked in either 5% goat serum or serum-free block (DAKO) for 30 min at RT. Antibodies were applied overnight at 4C: CC10, SP-C and TTF1 (Santa Cruz); BrdU (Serotec); p19Arf and Ki67 (Abcam); pT202/Y204-ERK1/2, p53 (Novocastra), p21 (Abcam) and p27KIP1 (CST). The appropriate secondary was applied followed either by an anti-peroxidase or ABC amplification for 3–60 minutes at room temperature. Antigen-antibody complexes were detected using a DAB chromagen system (Sigma). PBS with 0.1%(v/v) Tween 20 was used for all washes.
Protocol detail
Immunohistochemical Analysis of Lung Tissues
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Antibodies
Antigen antibody complexes
Brdu
Erk1
Goat
Lung
Methanol
P19arf
P27kip1
Peroxidase
Serum
Tissues
Ttf1
Tween 20
Corresponding Organization :
Other organizations : UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco
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Variable analysis
independent variables
- Preparation of lung tissue sections
- Quenching of endogenous peroxidase activity with 3%(v/v) H2O2 in methanol
- Blocking with 5% goat serum or serum-free block (DAKO)
- Application of antibodies (CC10, SP-C, TTF1, BrdU, p19Arf, Ki67, pT202/Y204-ERK1/2, p53, p21, p27KIP1) overnight at 4°C
- Application of appropriate secondary antibodies
- Antigen-antibody complex detection using a DAB chromagen system
- Time of incubation for blocking (30 min at RT)
- Time of incubation for secondary antibodies (3–60 minutes at room temperature)
- Use of PBS with 0.1%(v/v) Tween 20 for all washes
- Not explicitly mentioned
- Not explicitly mentioned
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