Following dimethylbenzene and rehydration with graded ethanol, antigen retrieval was performed using microwave irradiation.
After de-waxing in xylene and ethanol, antigen retrieval was achieved by boiling in a microwave oven for 5 min (pH 6.0, 0.94 ml Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA)/100 ml distilled water) three times.
Five micrometer thick paraffin-embedded sections were deparaffinized with xylene, rehydrated with a series of step down concentration of ethanol and antigen retrieval carried out with boiling 1x antigen retrieval buffer (BioGenex, San Ramon, CA).
After dewaxing in xylene and rehydration in ethanol for antigen retrieval, the slides were heated in a microwave oven in 0.02 M citrate buffer, pH 6.0.
After dewaxing in xylene and rehydration in ethanol, for antigen retrieval, the slides were placed in 0.02 M citrate buffer, pH 6.0 and boiled in the decloaking chamber.
The tissue sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol, then antigen retrieval was performed by boiling in citrate buffer (pH 6.0) for 30 min in the microwave oven.
After dewaxing in xylene and rehydration in decreasing ethanol concentrations, antigen retrieval was performed with 0.1 % protease pretreatment for 10 min at 37 °C (AppliChem, Darmstadt) for the detection of mCLCA5, mCLCA3 or club cell protein 10 and with microwave heating (600 W) in 10-mM citric acid (750 ml, pH 6.0) for 12 min for the detection of cytokeratin 5.
Briefly, the sections were deparaffinized in xylene and rehydrated in ethanol before enzymatic retrieval with Proteinase K (Dako Cytomation, Copenhagen, Denmark), followed by blocking with 4% bovine serum albumin and incubation with a mixture of TdT solution (enzyme solution) and labelled dUTP solution (label solution) in 37°C for 1 hour in a dark humified chamber.
The paraffin was removed by incubation in Histoclear (National Diagnostics, Atlanta, Georgia) and the section re-hydrated in ethanol before antigen retrieval by boiling in 10 mM citric acid pH 6 for 10 min. Sections were cooled, then washed in PBS before removal of endogenous peroxidase activity by incubation in 0.5% hydrogen peroxide/methanol for 30 min at room temperature.
The slides were deparaffinised in xylene and rehydrated in decreasing concentrations of ethanol, and antigen retrieval was performed in citrate buffer (pH 6.0) in a pressure cooker with the default program 125°C for 30 seconds followed by 90°C for 10 seconds at a pressure of 23 to 25 psi.
