Silane coated slides

A CCA tissue microarray (TMA, n = 354 and control, n = 2) was constructed by the Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. IHC reactions were performed on 4-µm-thick paraffin sections of CCA TMA on silane-coated slides (Sigma Chemical, USA) using the immunoperoxidase method. 16 The paraffin sections were de-waxed in xylene and rehydrated in serial dilution of ethanol. Antigen retrieval was performed in citrate buffer pH 6.0 under high pressure. Endogenous peroxidase activity was blocked in methanol with 3% hydrogen peroxide (HO). Non-specific binding was blocked by incubation in 5% normal horse serum, followed by overnight incubation at 4°C with 1:300 diluted rabbit anti-alpha 1 antitrypsin antibody (polyclonal, ab9373; Abcam, USA). Phosphate-buffered saline (PBS) was used instead of primary antibody in the control sections. Sections were washed with PBS and incubated with the specific secondary antibodies for 30 min at room temperature (1:300 diluted anti-rabbit horseradish peroxidase (HRP) conjugated, ab97051; Abcam). Slides were subsequently counterstained with Mayer's™ hematoxylin, dehydrated, cleaned, and mounted. All tissue slides were histopathologically evaluated by M.L., B.S., and the senior gastrointestinal pathologist, C.P.

Animals were anesthetized with 0.1% 3-aminobenzoic acid ethyl ester (MS-222, Sigma, St. Louis MO, USA) and killed by decapita-tion. After removal of the dorsal cranium, the heads were promptly immersed in a modified Bouin's fixative solution, consisting of a saturated aqueous solution of picric acid and formalin (ratio 3:1), for 24 h at room temperature.
After fixation and picric acid removal, the heads were decalcified in 0.25 M EDTA in 0.1 M phosphate buffer, pH 7.4, for 9 days at room temperature, dehydrated and then embedded in Paraplast Plus (Leica Biosystems, Richmond, IL, USA; melting point 55-57 • C). Frontal serial 5 m-thick sections were collected on silanecoated slides (Sigma). Adjacent slides were used for immunohistochemical detection.

IHC staining was performed using the DAKO LSAB kit (DAKO A/S, Glostrup, Denmark). Briefly, serial 4-µm-thick tissue sections were prepared on silane-coated slides (Sigma, St. Louis, MO, USA) and deparaffinized, rehydrated, and incubated with 3% H 2 O 2 in methanol for 15 min at room temperature. Subsequently, the antigen was retrieved in 0.01 M sodium citrate buffer (pH 6.0). The slides were incubated with rabbit anti-Mortalin antibody (ab53098; Abcam, Cambridge, MA, USA) at 4 °C overnight. After incubation with biotinylated secondary antibody at room temperature for 30 min, the slides were covered with streptavidin-peroxidase complex. IHC staining was developed using 3,3′-diaminobenzidine and counterstaining with Mayer's hematoxylin. We used liver sections as positive controls and rabbit IgG as an isotype control. Furthermore, the positive tissue sections were processed without primary antibody as a negative control.