Immediately after samples were removed from culture, they were placed in 4% paraformaldehyde for fixation for at least one week and then embedded in paraffin. Once all samples were in paraffin, they were sectioned along the sagittal plane and stained on the same day. Tissue were stained with hematoxylin and eosin for morphological analysis, α-smooth muscle actin α-SMA (clone 14A, Cell Marque) for myofibroblast formation and intercapsular adhesion28 (link)–30 (link), Ki-67 (clone 30–9, Ventana) for proliferation31 (link) and vimentin (clone V9, Ventana) a marker of both undifferentiated lens epithelium as well as differentiating fiber cells32 (link). All immunostainings were done with the BenchMark® ULTRA device (Ventana Medical Systems, Inc.). We also included negative immunostain controls to support the validity of our stains and identify possible experimental artefacts or background noise. These controls were processed in the same manner with the automated staining device but without the primary antibodies. Capsular adhesion, cellular morphology and the degree of staining of α-SMA, Ki-67 and vimentin were studied at the equatorial region of the capsule and at the center of the posterior capsule.
Benchmark ultra device
Manufactured by Roche
Sourced in United States
The BenchMark® ULTRA device is an automated immunohistochemistry and in situ hybridization (IHC/ISH) staining system. It is designed to streamline the staining process for clinical laboratories.
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Lab products found in correlation
5 protocols using benchmark ultra device
1
Histological Characterization of Lens Capsule
2
Histological Analysis of Intervertebral Disc Degeneration
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Directly after spinal dissection (baseline controls), or after the 14-day culture in the LDCS, IVDs were divided in halves; the left half was used for sagittal sectioning (a 3mm thick paramidsagittal tissue slices) and the right half was used for transverse sectioning (full thickness of IVD). Specimens were fixed in formaldehyde, decalcified using Kristensen’s solution (formic acid decalcifier buffered with formate), embedded in paraffin and 3-μm sections were cut with a microtome. Sections were stained with a standard H&E, alcian-blue staining and safranin-O staining. Histological classification of degeneration was performed according to the Rutges scale [36 (link)]. Baseline controls (day 0 IVDs) were used for region definition, sections of the SPL loaded IVDs were used as controls for the overloaded experimental groups.
Immunohistochemistry was performed using the fully automated Benchmark Ultra device (Ventana Medical Systems Inc., the Roche Group, Tucson, AZ, USA) with Optiview detective method. Cell apoptosis was detected using a cleaved Caspase-3 antibody (Cell Signaling Technology Inc. Danvers, MA, USA; rabbit poly clone, 9661-L, 1/100 dilution, 32 min incubation, 24 min retrieval). As a negative control we omitted the primary antibody, human tissue sections (healthy skin and cancerous trachea) were used for as positive controls.
Immunohistochemistry was performed using the fully automated Benchmark Ultra device (Ventana Medical Systems Inc., the Roche Group, Tucson, AZ, USA) with Optiview detective method. Cell apoptosis was detected using a cleaved Caspase-3 antibody (Cell Signaling Technology Inc. Danvers, MA, USA; rabbit poly clone, 9661-L, 1/100 dilution, 32 min incubation, 24 min retrieval). As a negative control we omitted the primary antibody, human tissue sections (healthy skin and cancerous trachea) were used for as positive controls.
3
Immunohistochemical Analysis of Hypoxia Markers
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Resected specimens were entirely formalin-fixed, and samples were mapped. For each tumour, one representative section was immunostained, and for each section, one tumour paraffin-embedded block was chosen for immunohistochemical analysis. Mapping, section and block selection were defined jointly by the same pathologist and nuclear physician for all patients based on PET data. Particular attention was paid to obtain the best correlation between immunohistochemistry and PET from the orientation of the surgical specimen to the section. Slides were immunostained using the following primary antibodies as hypoxia markers: Anti-HIF1-Alpha (1:500; ab8366; Abcam®, Cambridge, UK), anti-GLUT-1 (1:250), anti-CAIX (1:50), and anti-LDH-5 (1:1000). Deparaffinisation, antigen retrieval, and immunostaining were performed using a Benchmark ULTRA® device (Ventana-Roche®; Oro Valley, Arizona, USA). Slides were heated to 72 °C, incubated for 30 min with the antibody, and then rinsed in buffer solution. Primary antibody detection was performed using Ultraview DAB solution (Roche-Ventana®; Oro Valley, Arizona, USA). After rinsing, a drop of haematoxylin solution was applied within 4 min for counter-staining, before rinsing again. Finally, the slides were mounted with a glass coverslip.
4
Histological and Immunostaining Analysis
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The remaining halves of all samples, still in paraffin, were histologically sectioned at 5 µm using a microtome. They were then stained with hematoxylin and eosin (H&E) and immunostained with alpha smooth muscle actin (α-SMA) and Vimentin. Vimentin was immunolabeled using mouse monoclonal antibody V9 from Ventana, Oro Valley, AZ, USA) and α-SMA was immunolabeled using mouse monoclonal antibody 14A from Cell Marque, these immunostaining procedures were performed with the BenchMark® ULTRA device (Ventana Medical Systems, Inc.) following the manufacturers protocol and counterstained with Hematoxylin.
Samples were also stained with immunofluorescent wheat germ agglutinin (WGA) and 4′,6-diamidino-2-phenylindole (DAPI). Both stains were performed together by hand, WGA (L4895, Sigma-Aldrich, St. Louis, MO, USA) was applied at 10 μg/mL for 2 h and DAPI (MBD0015, Sigma-Aldrich) was applied at 1 μg/mL for 30 min. Fluorescent images were obtained with the Leica TCS-SP5 Confocal Microscope.
Samples were also stained with immunofluorescent wheat germ agglutinin (WGA) and 4′,6-diamidino-2-phenylindole (DAPI). Both stains were performed together by hand, WGA (L4895, Sigma-Aldrich, St. Louis, MO, USA) was applied at 10 μg/mL for 2 h and DAPI (MBD0015, Sigma-Aldrich) was applied at 1 μg/mL for 30 min. Fluorescent images were obtained with the Leica TCS-SP5 Confocal Microscope.
5
CXCL13 Expression in Renal Tumors
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All the hematoxylin-eosin stained samples belonging to the tumors were removed from the archive. Slides containing sufficient tumor and kidney parenchyma with normal histology were selected. Sections of four microns were taken from the paraffin blocks of these slides and stained with CXCL13 antibody (1:100 dilution, Genetex, USA) immunohistochemically in the Ventana Benchmark Ultra device.
While the tumor and intrarenal lymphocytes were evaluated in microscopic examination, the most intensely stained areas at low magnification were selected and CXCL13 positive staining cells were counted in ten high magnification areas. The H score for these cells was calculated with the formula = (3× strongly stained + 2× moderately stained + 1× weakly stained). For values between 0 and 300, the limit value was set as 40. Two groups were obtained as high and low staining, depending on whether it was above or below the cut-off value. In addition, staining of CXCL13 in tumor and kidney tissue was evaluated by dividing them into two groups in terms of the presence of staining.
The groups with low and high CXCL13 staining were compared with prognostic parameters, such as pT stage, WHO ISUP nuclear grade, tumor diameter, perirenal adipose tissue invasion, lymph node metastasis, distant metastasis, and risk factors, such as smoking and hypertension.
While the tumor and intrarenal lymphocytes were evaluated in microscopic examination, the most intensely stained areas at low magnification were selected and CXCL13 positive staining cells were counted in ten high magnification areas. The H score for these cells was calculated with the formula = (3× strongly stained + 2× moderately stained + 1× weakly stained). For values between 0 and 300, the limit value was set as 40. Two groups were obtained as high and low staining, depending on whether it was above or below the cut-off value. In addition, staining of CXCL13 in tumor and kidney tissue was evaluated by dividing them into two groups in terms of the presence of staining.
The groups with low and high CXCL13 staining were compared with prognostic parameters, such as pT stage, WHO ISUP nuclear grade, tumor diameter, perirenal adipose tissue invasion, lymph node metastasis, distant metastasis, and risk factors, such as smoking and hypertension.
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