Harris haematoxylin

For evaluation of in situ DPP4 activity, 10 µm frozen cryosections from ovarian tumours were air dried and incubated with 1.5 mM glycyl-prolyl-4-methoxy-β-naphthylamide and 2.5 mM Fast Blue BB Salt in assay buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20 at pH 7.6) for 30 min at 37 °C [24 (link)]. Sections were washed with assay buffer, nuclei were stained using Harris haematoxylin (Sigma-Aldrich, MO, USA) and slides were mounted with water. Slides were immediately imaged with a Nikon Eclipse 50i microscope (Nikon, Tokyo, Japan) using a 10× and 20× objective, and captured with a Nikon DS-Fi1 camera (Nikon, Tokyo, Japan). Ovarian tumour and liver sections treated with 1 µM sitagliptin (Januvia^®) served as negative controls, and liver sections served as positive controls. Images were analysed using ImageJ v1.0 (National Institute of Health, Rockville, MD, USA) for percentage of area and integrated density.

Harvested scaffolds from CAM were stained with haematoxylin and eosin (H&E) to observe vessel infiltration throughout the scaffold. Scaffolds were fixed overnight in 10% neutral buffered formalin solution at 4°C. Following fixation, scaffolds were dehydrated in increasing concentrations of ethanol until 100% ethanol was reached. Scaffolds were then placed within xylene solution under agitation, to dissolve out PCL filaments, after which they were processed using an automated tissue processor (ASP300, Leica, Germany) for paraffin infiltration. The samples were then embedded into paraffin blocks and were sectioned as described in Section 2.5 to obtain sections with a thickness of 7 μm in their cross-section to observe vessels infiltration. Sections were rehydrated in decreasing concentrations of ethanol, and then incubated with Harris haematoxylin (Sigma-Aldrich, Ireland) for 5 min before being washed in tap water for 5 min. Differentiation of the samples was then performed with acidified 70% ethanol, before being stained with 0.1% eosin Y (Sigma-Aldrich, Ireland) in 95% ethanol. Finally, slides were dehydrated and mounted with DPX. Images were captured as described in Section 2.5.

Tissues from the penetration experiments were fixed in 4% paraformaldehyde (Sigma) overnight. They were then cryopreserved in sucrose solutions at 10%, 20% and 30% for 6 h each and embedded in optimal cutting temperature (OCT) compound (Fisher Scientific, Loughborough, UK) and sectioned on a cryostat (Bright Instruments, Huntingdon, UK). In order to determine the integrity of the epithelial layers the slides were washed in 0.1 M phosphate buffered saline (PBS) and then stained with Harris Haematoxylin (Sigma), washed and differentiated in a 1% acid alcohol solution, washed in sodium bicarbonate for 2 min and finally counterstained in Eosin (Sigma). Sections were washed in tap water and dehydrated though a graded series of ethanols, cleared in Histoclear (National Diagnostics) and coverslips mounted using Vectamount (Vector Labs, Peterborough, UK) and then visualised using the Brightfield setting on an AxioPlan 2 microscope equipped with an AxioCam HRc camera and running Axiovision Software (all from Zeiss, Herfordshire, UK).

EGFR was assessed by immunohistochemistry, IHC, using a streptavidin-biotin complex technique as previously described.23 (link),24 (link) Endogenous peroxidase was blocked in 0.3% H₂O₂ in PBS for 20 min. Then, antigen retrieval was done in 0.05% protease K (Code no. S3020, Dako, Glostrup, Denmark) in PBS for 10 min at room temperature. The tissue sections were preincubated in PBS for 10 min and then the primary mouse monoclonal antibody (clone 31G7, Zymed labs, South San Francisco, CA, USA) directed against the EGF receptor, diluted 1:100, was added and the sections incubated overnight at 4°C. The secondary biotinylated antibody (goat anti-mouse, Dako, Glostrup, Denmark) and the peroxidase-labeled streptavidin-biotin complex (Dako, Glostrup, Denmark), diluted 1:200, were then added and the samples incubated for 30 min at room temperature. All sections were developed in 0.05% diamino benzidine (Sigma, St Louis, MO, USA) for 5 min and counterstained in Harris haematoxylin (Sigma, St Louis, MO, USA). Finally, the sections were dehydrated through graded alcohol to xylene and mounted in organic mounting medium (Pertex®, Histolab, Gothenburg, Sweden).

Hydrogels were fixed in 4% paraformaldehyde and processed for wax embedding. 6 µm-thick slices of each sample were cut, attached to a glass slide, dewaxed, rehydrated and stained as follows. Haematoxylin & eosin was used to visualize any nuclei still present after decellularization. Slides were stained with Harris Haematoxylin (Sigma-Aldrich) for 4 minutes, followed by a 10-minute wash with running tap water. Then slides were immersed in acid alcohol for 30 seconds and washed with tap water for 5 minutes. Finally, slides were stained with Eosin Y (Sigma-Aldrich) for 2 minutes. Picro-Sirius Red was used to assess collagen distribution. Slides were stained with Sirius Red (Sigma-Aldrich) in a saturated aqueous solution of picric acid for one hour and then for one minute in 0.5% acetic acid. Alcian blue was used to assess sGAG content. Slides were stained with 1% Alcian Blue 8GX (Sigma-Aldrich) in 0.1 M HCl for 5 minutes followed by three 30-second washes in dH₂O. After staining, all slides were dehydrated and coverslipped using DPX.

Sections adjacent to those used for DESI-MS were stained using haematoxylin and eosin (H&E). Following submersion in xylene (Sigma-Aldrich, UK) for 2 min, slides were immersed 4 times for 2 min in industrial methylated spirit (Sigma-Aldrich, UK). Following washing in tap water, sections were stained using Harris haematoxylin (3 min; Sigma-Aldrich, UK), rinsed in hot water, and dipped for one second in eosin (Leica, UK). Following further industrial methylated spirit (4 × 2 min) and xylene (4 × 2 min) washes, slides were left to dry. Samples were mounted with coverslips using aqueous mounting media (Abcam, UK) and left overnight to dry. Images were acquired using the 3D Histech Pannoramic 250 Flash II slide scanner with [20x/0.80 Plan Apo] objective and processed using Pannoramic viewer (3D HISTECH, Hungary).

Brown and Brenn (B&B) staining was done as previously described [10 (link)]. Slides were deparaffinized (xylene (Gadot, CAS 1330-20-7) 10 min twice) and rehydrated with descending concentrations of ethanol (100%—5 min, 90 and 70%—2 min each) to tap water (for 10 min). Thereafter, they were stained with a sequence of the following solutions; Harris haematoxylin (Sigma HHS32)—5 min, acid alcohol (1% HCl in 70% ethanol)—5 s, saturated lithium carbonate (Sigma 62470) in DDW—5 s, Hucker’s solution (0.2% crystal violet (sigma 61135)), 2% ethanol, 0.8% ammonium oxalate ((sigma 221716) in DDW)—2 min, iodine solution (0.33% iodine (BDH CAS 7553-56-2)), 0.66% potassium iodide ((J.T. Baker CAS 7681-11-0) in DDW)—15 s, ether acetone (50% Ether (Daejung CAS 60-29-7)), and 50% acetone (Gadot, CAS 67-64-1)—10 s, basic fuchsin (0.007% basic fuchsin (Sigma 857343, 7.31% ethanol in DDW))—3 min, acetone—10 s, picric acetone (0.1% picric acid in acetone EMS 26105-06)—20 s, acetone–xylene I (33.3% acetone, and 66.6% xylene)—5 s, acetone–xylene II (25% acetone, 75% xylene)—5 s, and xylene—1 min. The slides were mounted with entelane (mercury 1.07961) and covered with a coverslip. For analysis, each canal was divided into three parts, and evaluation was done by manually determining the most apical localization of bacteria in each canal. Statistics were performed using Fisher’s exact test.