The immunohistochemistry slides were scored by two pathologists (H.J.K and J.H.P). PD-L1 was scored both in tumor B-cells and in non-malignant immune cells. PD-L1 was considered positively expressed in tumor or non-malignant immune cells if membranous staining alone or membranous and cytoplasmic staining together was present. The percentage of stained tumor B-cells and non-malignant immune cells were estimated in each TMA core regardless of intensity. Tumor cells were distinguished from non-malignant immune cells by histologic clues such as nuclear enlargement and atypism, followed by comprehensive interpretation with other immunohistochemical markers including CD20, BCL2, BCL6, CD10 and MUM1. Since an optimal cut-off could not be determined by receiver operating characteristic (ROC) curve analysis, cases were classified by a 30% cutoff for tumor B-cells and 20% cutoff for non-malignant immune cells according to previous studies [6 (link)]. pSTAT3 expression was scored in tumor B-cells alone with cutoff value of 40%, which was set based on ROC curve analysis. In addition, the proportion (%) of pSTAT3-positive cells was digitally counted by using digital slide scanner and image analyzer (3DHISTECH, Budapest, Hungary) for correlation analysis.
Digital slide scanner
Manufactured by 3DHISTECH
Sourced in Hungary
The Digital slide scanner is a high-performance device designed for digitizing glass microscope slides. It captures high-resolution images of the entire slide surface, enabling efficient storage and analysis of microscopic samples.
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Lab products found in correlation
Horseradish peroxidase conjugated streptavidin biotin complex, by Agilent Technologies (6 mentions)
Mayer s hematoxylin, by Agilent Technologies (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Cm1950, by Leica camera (2 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Biosesang (1 mentions)
Quantcenter software, by 3DHISTECH (1 mentions)
α sma, by Agilent Technologies (1 mentions)
Abca1, by Novus Biologicals (1 mentions)
Anti pcna, by Santa Cruz Biotechnology (1 mentions)
Sirius red, by Chondrex (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Anti pcna antibody, by Santa Cruz Biotechnology (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Bcl 2, by Wuhan Servicebio Technology (1 mentions)
Fetal bovine serum (fbs), by ExCell Bio (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Calcein pi cell viability cytotoxicity assay kit, by Beyotime (1 mentions)
Propidium iodide, by Yeasen (1 mentions)
39 protocols using digital slide scanner
1
Immunohistochemical Analysis of PD-L1 and pSTAT3 in Lymphoma
2
Mast Cell and Serotonin Immunohistochemistry
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After dewaxing and antigen retrieval, the slides were incubated with 0.3% Triton X-100 for 30 min, and then with hydrogen peroxide for 10 min at room temperature. The slides were washed with PBST and blocked with goat serum for 30 min, and incubated with primary antibodies against mast cell tryptase (1:100) and 5-HT (1:100) overnight at 4 °C. Then, specific binding was detected by incubating with an HRP-conjugated secondary antibody and positive staining was visualized with a DAB substrate kit. Images were taken using a digital slide scanner (3DHISTECH, Budapest, Hungary).
3
Immunohistochemical Profiling of Inflammatory Markers
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After routine processing and blocking, sections were incubated at 4°C overnight with anti-mouse primary antibodies against tumor necrosis factor-alpha, TNF-α (RRID: AB_2835319), interferon-gamma, IFN-γ (RRID: AB_10857066), IL-17A (RRID: AB_2838094), IL-17F (RRID: AB_2842177), and IL-23(RRID: AB_10852886), or isotype control, respectively. After rinsing, sections were treated with Cy3-conjugated goat anti-rabbit IgG secondary antibody (RRID: AB_2861435) for 2 h, and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Image acquisition was performed with a digital slide scanner (3DHISTECH, Budapest, Hungary) under ECLIPSE TI-SR fluorescent microscope (NIKON, Tokyo, Japan). Positive immune cells and their values were determined to assess inflammatory changes.
4
Wound Tissue Analysis Protocol
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After monitoring the wound area, the mice were sacrificed, and the wound areas were collected and fixed with 4% paraformaldehyde (PFA, Biosesang, Seongnam-si, Republic of Korea). To remove residual PFA, the tissues were washed with PBS. After dehydration using several concentrations of alcohol, paraffin embedding was performed. Then, 4 μm-thick tissue slices were cut perpendicular to the wound surface, and the cut tissues were placed on a precoated slide with 0.1% w/v poly L-lysine (Sigma, St. Louis, MO, USA). To confirm regeneration of the wound area, hematoxylin and eosin staining was performed. Masson’s trichrome staining was performed to assess the degree of collagen synthesis. Tissue slides were scanned using a digital slide scanner (3D Histech, Budapest, Hungary) to analyze the tissue images as described in our previous report [50 (link)].
5
Quantitative Immunohistochemistry Analysis
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Consecutive FFPE sections were cut from each specimen. Primary antibodies, including anti-CD3 (D7A6E™, rabbit IgG, CST, 1:200), anti-45RO (UCHL1, mouse IgG2a, CST, 1:400), anti-CD8 (C8/144B, mouse IgG1, CST, 1:200), anti-CD11c (D3V1E, rabbit IgG, CST, 1:400), anti-Foxp3 (D2W83™, rabbit IgG, CST, 1:100), anti-CD20 (L26, mouse IgG2a, DAKO), anti-CD68 (KP1, mouse IgG1, DAKO) and anti-CD163 (10D6, mouse IgG1, Novus, 1:100) were incubated at 4 ℃ overnight. Then, the sections were incubated with secondary antibodies. Normal tonsil tissue was used as a positive control. Each stained slide was captured using a digital slide scanner (3DHISTECH, Budapest, Hungary) and analyzed by connected QuantCenter software (3DHISTECH, Budapest, Hungary). For IHC, the total number and density of cells expressing targeted proteins were quantified.
6
Carotid Stenosis Artery Tissue Analysis
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To analyze the expression of ABCA1, α-smooth muscle actin (SMA), and CD56 in carotid stenosis artery tissues, we used ABCA1 (Novus, Centennial, CO, USA), α-SMA (Dako, Santa Clara, CA, USA), and CD56 (Abcam, Cambridge, UK) antibodies, respectively. The arterial sections were incubated with 3% H2O2 in methanol to block endogenous peroxidase activity. After antigen retrieval, the slides were incubated with an anti-ABCA1, α-SMA, CD56 antibodies diluted at 1:100, 1:400, and 1:100 respectively at 4 °C overnight, and then incubated for 1 h with a biotinylated secondary anti-rabbit and mouse antibody at room temperature (RT). The sections were incubated with a horseradish peroxidase-conjugated streptavidin-biotin complex (Dako) and 3,3-diaminobenzidine (EnVision Systems, Santa Clara, CA, USA) was used to visualize the chromatic signals. Images were taken using a digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). Finally, the percentage of positive signals was quantified in all sections by 3DHISTECH Ltd. QuantCenter 2.2 program (3DHISTECH Ltd.).
7
Quantifying Hepatocyte Proliferation after MSC Transplantation
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To observe the degree of hepatocyte proliferation following transplantation with MSCs or the control, BDL rat liver tissues were stained with anti-PCNA (Santa CruzBiotechnology, Dallas, Texas, USA) using immunohistochemistry. The liver tissues were embedded in paraffin and sectioned. The sectioned tissues were incubated in 3% H2O2 in methanol to block endogenous peroxidase activity. After antigen retrieval, the tissues were incubated with a primary antibody (1:200) at 4 °C overnight, followed by a 1-h incubation with biotinylated secondary anti-rabbit antibody at room temperature. Incubation with horseradish peroxidase-conjugated streptavidin–biotin complex (DAKO, Santa Clara, CA, USA) and 3,3-diaminobenzidine (EnVision Systems, Santa Clara, CA, USA) was performed to generate a chromatic signal. The samples were counterstained with Mayer’s hematoxylin (DAKO). Additionally, the percentage of hepatocytes with PCNA-positive nuclei relative to the total number of hepatocytes was calculated in randomly selected sections using a digital slide scanner (3DHISTECHLtd., Budapest, Hungary). The experiment was analyzed in at least triplicate.
8
Coumarin-6 for Skin Penetration Analysis
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Coumarin-6 was used as a fluorescent probe to investigate the penetration of the carriers in the porcine ear skin. In vitro skin penetration of the coumarin-6-loaded transfersomes (Cou6-Ts) and coumarin-6-loaded L-cysteine modified transfersomes (Cou6-LCTs) was carried out using a Franze diffusion cell. At 3 h and 8 h, the skin samples were collected and washed with physiological saline to remove the residual formulation on the skin surface. The treated skin was successively cut off and frozen at −80 °C, and then the cross-section perpendicular to the skin surface and the horizontal cross-section parallel to the skin surface were prepared using a freezing slicer (Leica CM 1950, Nussloch, Germany). DAPI was used for nuclear staining of the tissue sections, and fluorescence images were collected using a digital slide scanner (3Dhistech, Budapest, Hungary) to observe the fluorescence distribution in porcine ear skin.
9
Quantifying Hepatic Collagen Deposition
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According to the manufacturer’s instruction, the manufacturer’s instruction measured collagen deposition in liver sections in Sirius Red (Chondrex, USA). To analyze hepatic collagen distribution, fibrotic septa randomly selected from the right and left liver lobes of 6 individual mice/groups were assessed. Images were acquired using a digital slide scanner (3DHISTECH, Hungary). Collagen extent was expressed as a percentage of stained area in each liver section.
10
Hepatocyte Proliferation Analysis
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To analyze the degree of hepatocyte proliferation in tissues following treatment with WKYMVm or no treatment, we used an anti-proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Dallas, TX, USA) antibody. The liver sections were incubated with 3% H2O2 in methanol to block endogenous peroxidase activity. After antigen retrieval, the slides were incubated with an anti-PCNA antibody diluted 1:200 at 4 °C overnight, and then incubated for 30 min with a biotinylated secondary anti-rabbit antibody at room temperature (RT). The sections were incubated with a horseradish peroxidase-conjugated streptavidin-biotin complex (Dako, Santa Clara, CA, USA) and 3,3-diaminobenzidine (EnVision Systems, Santa Clara, CA, USA) to visualize the chromatic signals. Images were taken using a digital slide scanner (3DHISTECH Ltd., Budapest, Hungary). Finally, the percentage of PCNA-positive hepatocytes was quantified in randomly selected sections (three fields per group at 400× magnification).
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