Deidentified human liver tissue from patients diagnosed with late-stage PSC were collected by Dr. Burcin Esker at Indiana University School of Medicine following written informed consent and IRB approval. Liver samples were processed, embedded into paraffin blocks, and sectioned at 5 μm for staining. Immunohistochemistry staining of bile ducts using anti-CK-19 antibody (Abcam; Waltham, MA), Vector ImmPRESS® HRP Reagent Kit and chromogen development using the Vector ImmPACT® DAB substrate (Vector Laboratories; Burlingame, CA) was performed. FGSR staining solution is made by adding Picrosirius Red (0.1%) and Fast Green FCF (0.1%) to saturated picric acid (1.2% w/v), and slides are incubated in the pre-made solution for 30 min. We chose to use FGSR staining since it provides well-defined collagen fibers and the highest sensitivity for quantitative analysis compared to Sirius Red alone [8 (link)]. Lastly, slides are cleared, mounted, scanned using the Lecia Aperio AT2 system and imaged with the Lecia Aperio ImageScope software (Leica Biosystems; Buffalo Grove, IL).
Vector immpact dab substrate
Manufactured by Vector Laboratories
Sourced in United Kingdom
Vector ImmPACT® DAB substrate is a chromogenic substrate used for the detection of horseradish peroxidase (HRP) in immunohistochemistry and other applications. It produces a brown reaction product upon enzymatic conversion.
Automatically generated - may contain errors
2 protocols using vector immpact dab substrate
1
Histological Assessment of Late-Stage PSC
2
Histological Analysis of Xenografts
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Xenografts of MDA-MB-231 were collected from previous published experiments [9 (link)]. The tumors were fixed in 4% paraformaldehyde overnight at room temperature, and subsequently, 6 μm paraffin-embedded sections were used for staining. For histological examination, the sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, the above described primary antibodies for immunofluorescence were used. After incubation with biotinylated secondary antibodies and horse radish peroxidase (Vectastain® Elite ABC Reagent, Vector, Peterborough, UK), diaminobenzidine (Vector ImmPACT DAB substrate, Vector, Peterborough, UK) was applied for 5 min before mounting in Permount (Fisher Scientific, Hampton, NH, USA). The sections were subsequently imaged using a Nikon eclipse 50i microscope and a Qimaging micropublisher 5.0 rtv camera.
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