Around 4-µm-thick FFPE sections were used for manual staining. Briefly, FFPE sections were deparaffinized in xylene-1 and xylene-2 for 5 min each, rehydrated in graded alcohol (100% ethanol, 90% ethanol, 70% ethanol, and 50% ethanol), dipping slides for 2 min in each. Slides were immersed in tris-ethylenediaminetetraacetic acid (EDTA) buffer (pH 9.0), heated in a domestic microwave oven at full power three times each for 5 min and left in buffer to cool at room temperature for at least 20 min. The sections were then incubated in 3% hydrogen peroxide (H2O2) for 30 min at room temperature to block endogenous peroxidase activity. Then placed in protein blocking for 20 min, and incubated with the primary antibody (anti-VDR) at dilution 1:4,000 in 1% bovine serum albumin/tris-buffered saline overnight in a humid chamber at 4 °C.
The following day, the slides were washed and incubated with 100 µL of biotinylated secondary antibody (SignalStain® Boost IHC Detection Reagent; Cell Signaling Technology) for 30 min in a humidified slide tray, then with 100 µL of streptavidin peroxidase at room temperature for 20 min. 3,3’-diaminobenzidine (DAB) was used following the manufacturer’s instructions (HRP kit; Cat# ab64264). Slides were dried on the tissue and kept for 4 min on a humidified slide tray, washed with distilled water for 5 min and counterstained with pre-diluted hematoxylin stain for 2 min and rinsed with running tap water. Dehydration with four series of graded alcohol (70% ethanol, 80% ethanol, 90% ethanol and absolute ethanol) by dipping slides for 5 min each, then cleared in two series of xylene for 5 min each. Lastly, slides were mounted with di-N-butyl phthalate in xylene (DPX) and air-dried before microscope examination. A benign BC slide was used as a control for anti-vitamin D antibody.
The following day, the slides were washed and incubated with 100 µL of biotinylated secondary antibody (SignalStain® Boost IHC Detection Reagent; Cell Signaling Technology) for 30 min in a humidified slide tray, then with 100 µL of streptavidin peroxidase at room temperature for 20 min. 3,3’-diaminobenzidine (DAB) was used following the manufacturer’s instructions (HRP kit; Cat# ab64264). Slides were dried on the tissue and kept for 4 min on a humidified slide tray, washed with distilled water for 5 min and counterstained with pre-diluted hematoxylin stain for 2 min and rinsed with running tap water. Dehydration with four series of graded alcohol (70% ethanol, 80% ethanol, 90% ethanol and absolute ethanol) by dipping slides for 5 min each, then cleared in two series of xylene for 5 min each. Lastly, slides were mounted with di-N-butyl phthalate in xylene (DPX) and air-dried before microscope examination. A benign BC slide was used as a control for anti-vitamin D antibody.
