At 2 weeks after co-culturing hAC and hOB on the scaffolds, they were fixed in 3.7% paraformaldehyde solution for 3 h, washed with PBS, embedded in paraffin, and sectioned with a microtome (HistoCore Multicut microtome equipment, Leica Biosystems) at a thickness of 10 µm. Then, the sections were floated in a water bath at 40 °C, positioned on poly-L-lysine-coated microscope slides, baked overnight at 37 °C, stained with hematoxylin and eosin (H&E), and images recorded with a Nikon Eclipse E600 microscope with an attached Nikon DS-Fi1-L2 camera. Processing, staining, and mounting of samples were performed with a BenchMark Ultra IHC/ISH, and VENTANA HE 600 systems (Roche Tissue Diagnostics). In vitro histological evaluations were performed on the processed samples.
Ventana he 600
Manufactured by Roche
Sourced in United States, Switzerland
The Ventana HE 600 is a slide stainer used in histology laboratories. The device automatically applies reagents to tissue samples mounted on microscope slides, facilitating the preparation of specimens for microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Dm1000, by Leica (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Optiview dab ihc detection kit, by Roche (1 mentions)
Ultraview universal alkaline phosphatase red detection kit, by Roche (1 mentions)
Ab859, by Abcam (1 mentions)
B0586, by Agilent Technologies (1 mentions)
Ventana discovery ultra, by Roche (1 mentions)
Ventana discovery xt, by Roche (1 mentions)
Tissue tek vip, by Sakura Finetek (1 mentions)
8 protocols using ventana he 600
1
Histological Analysis of Co-cultured hAC and hOB
2
Pathological Evaluation of Resection Margins
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The resected specimens were processed and fixed immediately, before the lesion size was measured. After fixation with 10% formalin (20-25˚C for 12-24 h), the specimens were sent for pathological examination. The samples were cut into continuous sections at 2-mm intervals from top to bottom and embedded in paraffin. In total, three sections were prepared from each tissue and then stained with hematoxylin and eosin using the Ventana HE 600 automatic staining machine (Roche Diagnostics; 20-25˚C). An experienced pathologist (Dr Yao Liu, Department of Pathology, Fourth Hospital of Hebei Medical University) then used a light microscope (DM1000; Leica Microsystems GmbH; magnification, x200) to evaluate tumor involvement in the lateral or deep resection margin to avoid misjudgment. When examining the tissue sections for pathological evaluation, all sections were observed from the top of the tissue to identify the nature of the lesion, the degree of tumor differentiation, whether the vertical/transverse edge of the tumor was positive and whether there was vascular infiltration. A positive resection margin as defined as the presence of atypical cells (LIN, HIN or invasive cancer) at the lateral or deep resection margin.
3
Automated H&E Staining on Ventana HE600
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H&E staining was executed in a fully automated manner following the standard protocol on the Ventana HE600 stainer (Roche Tissue Diagnostics).
4
Immunohistochemical Staining of SOX10 in Melanoma
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From each tissue block, one paraffin section of 3 µm was cut and mounted on a Superfrost Plus slide (Thermo Fisher Scientific, Waltham, MA, USA). They were dried at 60 °C for 1 h. H&E stains were performed by Ventana HE 600 (Roche Diagnostics, Tucson, AZ, USA) and IHC by Ventana Benchmark Ultra (Roche Diagnostics). SOX10 IHC positivity was visualized with the SOX-10 Rabbit Monoclonal Primary Antibody (SP267; ready-to-use; 32 min; Roche Diagnostics) in combination with either the OptiView DAB IHC Detection Kit (Roche Diagnostics; brown chromogen) or the ultraView Universal Alkaline Phosphatase Red Detection Kit (Roche Diagnostics; red chromogen). Standard settings and regent kits of Ventana Benchmark Ultra (Roche Diagnostics) were used for antigen retrieval (Cc1, 32 min) and endogenous peroxidase blocking (only DAB stains). Immunohistochemical slides were counterstained with Mayer’s hematoxylin and bluing reagent. Internal controls were present in primary melanomas (SOX10 positivity in epidermis).
5
Tissue Microarray Construction and Staining
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The sections were carefully reviewed, and representative tumor regions were selected as regions of interest for the study. Markers were made on HE section slides for subsequent preparation of tissue microarrays by punching the corresponding tissue wax blocks. Tissue microarrays were prepared with a manual tissue microarray sampling gun (JLM-5113, Guangdong, China). The tissue columns were incorporated into 60-well TMA wax molds and paraffin-embedded. Then, the sections of the TMA (2 µm) were transferred to detachable slides, and HE staining was performed by applying an automatic stainer (Ventana HE600, Roche, USA).
6
Immunohistochemical Analysis of HBV Antigens
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Formalin-fixed paraffin-embedded livers were sectioned at 5 µm thickness, mounted on pre-charged slides and stained with HBcAg (B0586, Dako, Glostrup, Denmark) and HBsAg (ab859, Abcam, Cambridge, UK). Tissues were subjected to an EDTA-based antigen retrieval while signal amplification and detection were achieved with a hapten multimer and a DAB chromogenic detection kit on the Ventana Discovery Ultra autostainer (Roche Diagnostics, Rotkreuz, Switzerland). Negative control staining was performed for every antibody using an isotype control (Rabbit PE IgG for HBcAg, mouse IgG1 for HBsAg). Sections were coverslipped with the automated Ventana HE600 (Roche Diagnostics, Rotkreuz, Switzerland) and scanned using the Hamamatsu Nanozoomer RX.
7
Immunohistochemical Evaluation of FAP Expression
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Hematoxylin-eosin (H&E) slide staining was conducted at a routine laboratory of the Institute of Pathology, University Hospital Essen, using the automated Stainer Ventana HE 600 (Roche, Basel, Switzerland). Immunohistochemical staining was performed according to our routine standard protocols) using an automated Stainer (Ventana Discovery XT). FAP was stained using the SP325 monoclonal antibody (Abcam, 48 min antigen retrieval in buffer with 60 min incubation time, dilution 1:100).
FAP expression was evaluated using the immune-reactive score (IRS). The overall percentage of the primary tumour with stroma FAP staining was evaluated semi-quantitatively (0, 1+, 2+, 3+), as well as the staining intensity (none, weak, moderate, strong) [11 (link)]. The IRS is composed of the number of positively stained cells (percentage point (PP)) and the staining intensity (IS). Both parameters, multiplied, result in the IRS (PP × SI) [12 (link)].
FAP expression was evaluated using the immune-reactive score (IRS). The overall percentage of the primary tumour with stroma FAP staining was evaluated semi-quantitatively (0, 1+, 2+, 3+), as well as the staining intensity (none, weak, moderate, strong) [11 (link)]. The IRS is composed of the number of positively stained cells (percentage point (PP)) and the staining intensity (IS). Both parameters, multiplied, result in the IRS (PP × SI) [12 (link)].
8
Automated Tissue Processing and Staining
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Tissue processing was performed with an automated program performed on a Tissue-Tek VIP instrument (Sakura Finetek USA, Inc., Torrance, CA, USA) utilizing an aqueous-free pathway for PAXgene-fixed samples, resulting in the creation of PFPE and FFPE tissue blocks in a standard fashion. A representative 5-micrometer section was obtained from each and stained with Hematoxylin and Eosin on a Ventana HE 600 automated staining system (Roche Diagnostics, Basel, Switzerland) according to a standard laboratory protocol.
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