Fast red

The miRCURY LNA™ microRNA Detection Kit (QIAGEN, Hilden, Germany) was used to detect the expression of miRs in PDAC tissue sections according to the instructions of the manufacturer. The hybridization was performed for 2 h at 54 °C using a specific 3′,5′ digoxigenin (DIG)‑labeled LNA miR detection probe. A scrambled, DIG-labeled miR served as a negative control. The sequences of the miR probes were as follows: hsa-miR-378i: 5′-DiGN-CCTTCTGACTCCTAGTCCAGT-3′-DiGN_N; hsa-miR-378a-3p: 5′-DiGN-CCTTCTGACTCCAAGTCCAGT-3′-DiGN_N. The bound miR probes were detected with nitroblue tetrazolium/5-bromo-4-chloro-30-indolyl phosphate p-toluidine (NBT/BCIP; Vector Laboratories, Burlingame, CA, USA), which served as a substrate. For nuclear staining, patient tissue slices were incubated in Fast Red (Vector Laboratories, Burlingame, CA, USA). Positive signals in ten randomly selected fields were counted blindly by two examiners.

As described [25 (link)], ovarian samples were first fixed in 2% paraformaldehyde-0.2% glutaraldehyde in a 0.1 M phosphate buffer solution (pH 7.4), and then stained with X-gal staining buffer containing 1 mg/ml X-gal, 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide. After the staining procedure, samples were post-fixed with 10% neutral buffered formalin for histological analysis. Fast red (Vector Laboratories) was used to counterstain the nucleus.

Immunohistochemical staining was carried out using the VECTASTAIN ABC Kit (Vector Laboratories, Burlingame, CA, USA) in accordance with the manufacturer's instructions. Samples were treated with 0.5% H₂O₂ to block endogenous peroxidase activity for 10 min and permeabilized with 0.3% Triton‐X100. Non‐specific binding was inhibited by incubating samples with 1% normal horse serum for 1 h at room temperature (RT). Primary mouse anti‐CD31, anti‐F4/80 and anti‐SDF‐1α antibodies (Abcam, Cambridge, MA, USA) were added. After three PBS washes, samples were incubated with a biotin‐conjugated secondary antibody for 1 h at RT. After washing with PBS, the substrate solution was added and the reaction was allowed to proceed at RT for 40 min. To visualize the reactive area in the tissue, samples were treated with dimethyl‐aminoazobenzene (DAB; Vector Laboratories), counterstained with Fast Red (Vector Laboratories) for 10 min and mounted. Finally, samples were observed using a Nuance Multiplex Biomarker Imaging System (Cambridge Research Instrumentation, Woburn, MA, USA).

In brief, paraffin-embedded tissue blocks of the SN were cut into 6 μm-thick consecutive sections with a microtome and incubated with mouse-anti tyrosine hydroxylase (TH, dilution 1:800 in TBS-Trition 0.1%, Immunostar, Hudson, USA) at 4°C overnight. TH immunoreactivity was visualized with Vector SG grey (Vector, California, United State) followed by counter-staining with fast red (Vector, California, United State), and dehydrated in a series of ethanol, xylene, then mounted with Entellan.

The pellets were fixed in 4% formaldehyde for 2 h, dehydrated, and paraffin-embedded. Sections of 5 µm thickness were deparaffinized, rehydrated, stained with Safranin O (0.2% in 1% acetic acid; Fluka, Sigma-Aldrich, St. Louis, MO, USA), and counterstained with Fast Green (0.04% in 0.2% acetic acid; Merck Millipore, Darmstadt, Germany) according to standard histological protocols. Immunohistological staining of type II collagen was performed as described previously [2 (link)]. In brief, sections were treated with 4 mg/mL hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) followed by 1 mg/mL pronase (Roche Diagnostics, Basel, Switzerland), and unspecific binding sites were blocked with 5% bovine serum albumin. Type II collagen was detected with a monoclonal mouse anti-human type II collagen antibody (1:1000, 0863171-CF, clone II-4C11, MP Biomedicals, Santa Ana, CA, USA) and visualized with biotinylated goat anti-mouse secondary antibody (1:500, Dianova, BIOZOL, Hamburg, Germany), streptavidin-alkaline phosphatase (Dako, Agilent, Santa Clara, CA, USA), and Fast Red (Vector Laboratories, Newark, CA, USA).

Femurs were isolated and fixed in 3.7% formaldehyde (Sigma-Aldrich). Samples were decalcified with decalcifying solution (Sigma-Aldrich) and processed with a TP1020 tissue processor (Leica Biosystems, Wetzlar, Germany) to prepare paraffin blocks, and 5.0 μm thick sections were prepared. For trichrome staining, a NovaUltraTM Masson trichrome stain kit (IHC World, Woodstock, MD, USA) was used. For immunohistochemistry staining, the VECTASTAIN ABC kit or ABC-AP kit (Vector Laboratories, Burlingame, CA, USA) was used. Briefly, rehydrated samples were boiled with 0.01 M sodium citrate (Sigma-Aldrich) for antigen retrieval. For blocking activity of endogenous hydrogen peroxidase, the samples were treated with 0.5% H₂O₂. Next, the samples were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich). For blocking nonspecific binding of antibodies, the samples were incubated with 2% normal horse serum for 1 h at room temperature (RT) and then treated with primary antibodies against CD31, endomucin. The samples were incubated with biotinylated secondary antibodies for 1 h at RT, followed by incubation with an avidin–biotin complex solution. The substrate solution, ImmPACT NovaRed or Vector Blue (Vector Laboratories), was used to visualize the reactive area in the tissue. Finally, samples were counterstained with Fast Red (Vector Laboratories).