Scn400 scanner

Homozygous mice (Trpc1^lacZ) were anesthetized and transcardially perfused with 4% paraformaldehyde in PBS. Brains were isolated, cryoprotected, and frozen sectioned (16 μm) in sagittal plane. For β-galactosidase activity detection, sections were fixed in 2% glutaraldehyde, washed, and stained 72 h at 37°C in PBS supplemented with 20 mM K₃Fe(CN), 20 mM K₄Fe(CN)₆, 2 mM MgCl₂, and 1 mg/mL X-gal. They were then washed and counterstained with Nuclear Fast Red. No staining was detected on sections from wild-type mice treated in the same conditions. A Leica SCN400 scanner acquired the pictures of complete sagittal brain sections.

In order to measure the amount of IBDM in wildtype and MDR2KO mice in the absence or presence of corticosterone, IHC on paraffin-embedded liver sections was performed using a CK19 antibody for labeling the cholangiocytes, followed by VectaStain ABC kit from Vector Laboratories Inc. (Burlingame, CA, USA) for color development, following previous described protocols [9 (link)]. Liver sections of 4 μm were obtained with a Leica microtome. The VectaStain-stained sections were mounted on slides, and scanned with a Leica SCN400 scanner, with 20× optical magnification. Screenshots with 10× digital magnification were taken and used for quantification of field-stained pixel percentage of CK19-positive cells. The same procedure was employed for IHC of CD68, F4/80, Clec4f, Ccr2 and αSMA in liver sections. The images were analyzed with ImageJ software downloaded from the NIH website. The degree of fibrosis in FVBN and MDR2KO mice treated with vehicle or corticosterone was assessed by staining collagen I and III fibers in liver sections with Sirius Red (kit from Sigma Aldrich, St. Louis, MO, USA) and then processing the slides in the same manner used for IHC analysis. Apoptosis in the liver of FVBN and MDR2KO mice was assessed using a TUNEL kit from Abcam (Cambridge, MA, USA) according to manufacturer’s instructions and was conterstained with methyl green.