The primary antibodies used included VK9, a monoclonal antibody against Globo H (hybridoma generously provided by Dr. Govindaswami Ragupathi (Memorial Sloan‐Kettering Cancer Center, New York, NY) and anti‐Ki‐67 (NCL‐L‐Ki67‐MM1; Leica Biosystems, Concord, Canada). Globo H immunohistochemical (IHC) analysis was performed on FFPE human and rat ICC tissues as described previously.(20 ) Details of IHC methods were described in the Supporting Information .
Ncl l ki67 mm1
Manufactured by Leica
Sourced in United Kingdom, Germany
The NCL-L-Ki67-MM1 is a monoclonal antibody used for the immunohistochemical detection of the Ki-67 protein, which is a marker of cellular proliferation. This product is intended for in vitro diagnostic use.
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Lab products found in correlation
Horseradish peroxidase conjugated streptavidin, by Vector Laboratories (1 mentions)
Dab substrate chromogen solution, by Agilent Technologies (1 mentions)
Bz x710, by Keyence (1 mentions)
Ncl l mitf, by Leica (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Tcs spe, by Leica (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Ab2413, by Abcam (1 mentions)
M0814, by Agilent Technologies (1 mentions)
M0823, by Agilent Technologies (1 mentions)
Cx 294, by Agilent Technologies (1 mentions)
9 protocols using ncl l ki67 mm1
1
Globo H Immunohistochemistry Analysis
2
Immunohistochemical Analysis of Puf-A, p53, and Ki-67
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Formalin-fixed paraffin sections were incubated overnight with antibodies against Puf-A, p53 (NCL-P53-DO7, Novocastra, Leica Biosystems, UK), and Ki-67 (NCL-L-Ki-67-MM1, Leica Biosystems). After washing, tissue slides were incubated with biotinylated secondary antibodies for 1 h at room temperature, and then incubated with horseradish peroxidase-conjugated streptavidin (Vector, USA). All sections were counterstained with Mayer’s hematoxylin. Puf-A, p53, and Ki-67 stained tissue sections were read by the pathologist YLH and evaluated with a histological scoring system, H-score [49 (link)]. Ki-67 was low if there is <14% of nuclei staining and high if ≥14% [50 (link), 51 (link)]. The Youden index, which was calculated from the receiver operating characteristic curve, was used to determine the optimal cut-off value for high versus low expression level.
3
Immunohistochemical Analysis of Mucins and Markers
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Immunohistochemical analysis was performed with a Dako Envision + Mouse/Rabbit Peroxidase Detection System (Dako Cytomation, Carpinteria, CA, USA). Antigen retrieval was performed using a pressure cooker heated to 100 °C in citrate buffer (pH 6.0) for 5 min. Peroxidase activity was blocked with 3% H2O2-methanol for 10 min.
Sections were incubated with anti-MUC5AC (anti-MRQ-19, 760-4389, V0001348, Roche; 1:2 dilution for IHC), anti-MUC6 (NCL-MUC-6, 6014968, Leica Biosystems; 1:100 dilution for IHC), anti-MUC2 (anti-MRQ-18, 760-4388, V0001436, Roche; 1:2 dilution for IHC), anti-CD10 (56C6, CD10-270, 602420, Leica Biosystems; 1:100 dilution for IHC), or anti-Ki67 (NCL-L-Ki67-MM1, Leica Biosystems; 1:100 dilution for IHC) antibodies for 1 h at 25 °C, followed by incubation with Envision and the anti-mouse peroxidase for 1 h at room temperature. For color reactions, sections were incubated with DAB substrate-chromogen solution (Dako Cytomation) for 5 min. Sections were counterstained with 0.1% hematoxylin & eosin. Reactions lacking a primary antibody were used as negative controls.
Sections were incubated with anti-MUC5AC (anti-MRQ-19, 760-4389, V0001348, Roche; 1:2 dilution for IHC), anti-MUC6 (NCL-MUC-6, 6014968, Leica Biosystems; 1:100 dilution for IHC), anti-MUC2 (anti-MRQ-18, 760-4388, V0001436, Roche; 1:2 dilution for IHC), anti-CD10 (56C6, CD10-270, 602420, Leica Biosystems; 1:100 dilution for IHC), or anti-Ki67 (NCL-L-Ki67-MM1, Leica Biosystems; 1:100 dilution for IHC) antibodies for 1 h at 25 °C, followed by incubation with Envision and the anti-mouse peroxidase for 1 h at room temperature. For color reactions, sections were incubated with DAB substrate-chromogen solution (Dako Cytomation) for 5 min. Sections were counterstained with 0.1% hematoxylin & eosin. Reactions lacking a primary antibody were used as negative controls.
4
Quantification of Melanocytes in FFPE Skin
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Formalin-fixed paraffin-embedded (FFPE) human skin tissue sections from organotypic tissue were stained for MITF (NCL-L-MITF, Leica Biosystems, Nussloch, Germany), MelanA (NCL-L-MITF, Leica Biosystems), and Ki67 (NCL-L-Ki67-MM1, Leica Biosystems). Staining was performed following the manufacturer’s protocol for high-temperature antigen unmasking technique for paraffin sections. For melanin staining, FFPE tissue was subjected to Fontana-Masson histochemical stain as previously described (21 (link), 22 (link)). Tissue section quantification was performed according to previous reports (22 (link)). Briefly, 10× photomicrograph images of representative tissue sections were taken using Keyence BZ-X710 (Itasca, IL, USA). Tiff files of the images were saved and transferred to FIJI (ImageJ). Images corresponding to the single specific color were then analyzed to determine the number of pixels in each sample and normalized to epidermal area. The numbers of pixels representing MelanA staining were normalized to the total amount of epidermal area.
5
Proliferative Analysis of Cardiomyocytes
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To determine proliferative active CMCs, paraffin sections were incubated with a mixture (1:1) of primary antibodies for Ki67 (1:200, rabbit anti-human, NCL-L-Ki67-MM1, Leica Biosystems, Buffalo Grove, IL, USA) and sarcomeric α-actin (Sarc α-act; 1:50, mouse anti-human, Abcam, Cambridge, UK), and then with a mixture (1:1) of secondary antibodies labeled with fluorochromes Alexa 488 (1:200, chicken anti-rabbit, Thermo-Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 546 (1:200, donkey anti-mouse, Thermo-Fisher Scientific, USA). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Merck KGaA, Darmstadt, Germany). The preparations were examined using a confocal laser microscope Leica TCS SPE (Leica Microsystems Gmbh, Austria). The amount of Ki67+/Sarc α-act+ CMCs was counted. The area of the section was measured and the density of the CMCs was determined. The proportion of Ki-67+ CMCs/106 CMCs in the myocardium was determined (median, min–max).
6
Quantification of Cell Proliferation and Apoptosis
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Detailed methods are provided in the online version of this paper and include the following: were incubated with primary antibody overnight at 4 C, washed 3 times with PBS and incubated with secondary antibody for 1 h at room temperature followed by washing 3 times with PBS. All slides were incubated with mounting medium containing DAPI (Solarbio, Cat #C0060) for 5 min. Primary antibodies for immunofluorescence staining included: a-CD68 (1:50 dilution, Biolegend, Cat # 137002, clone FA-11), a-Cleaved Caspase 3 (1:100 dilution, Cell Signaling Technology, Cat # 9664S, clone 5A1E), a-Ki67 (1:100 dilution, Leica Biosystem, Cat # NCL-L-Ki67-MM1, clone MM1). Secondary antibodies used in these experiments were included as following: Alexa Fluor 488 Goat anti-mouse IgG (H + L) (1:200 dilution, Invitrogen, Cat # A-11029), Alexa Fluor 488 donkey antirabbit IgG (H + L) (1:200 dilution, Life Technologies, Cat # A-21206), Dylight 649 Goat Anti-Rat IgG (H + L) (1:200 dilution, EarthOx, Cat #E032640-01). Images were captured by Zeiss LSM710 (AXIOobserze.Z1). Three independent immunofluorescent images from each sample covering tumor areas were taken. The Ki-67 + and Cleaved Caspase 3 + cells in each image was manually counted and the average percentage of respective positive cells were calculated against DAPI + cells.
7
Pancreatic Neuroendocrine Tumor Grading
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All patients included in this study underwent surgery for localized or metastatic disease. The Ki67 evaluation was expressed as a percentage based on the count of Ki67-positive cells within the tumour, using NCL-L-Ki67-MM1 (Novocastra, Newcastle Upon Tyne, UK) and KI67 CL. 30-9 (Ventana Medical System Inc., Tucson, ARI, USA), antibodies; the spot with the highest immunostaining was considered when intratumoural heterogeneity was present. The 2017 WHO classification was applied to determine tumour grade, and tumours were classified as PanNET G1 (Ki67 < 3%) or PanNET-G2 (Ki67 3–20%) or PanNEN-G3 (Ki-67 > 20%). Patients with poorly differentiated lesions with Ki67 > 20% were classified as PanNEC.
8
Immunohistochemical Analysis of Tissue Sections
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Tissue sections were fixed in 4% buffered formaldehyde, embedded in paraffin, cut at 3 µm and stained by hematoxylin eosin or used for immunostaining. Immunohistochemistry was performed for keratin 14 (K14), laminin and the proliferation marker Ki-67. K14 was labeled with a guinea pig antibody (k14.2; Progen, Heidelberg, Germany) at a dilution of 1∶100 and laminin was labeled with a 1∶50 dilution of rabbit polyclonal antibody donated by Dr. Stark, DKFZ, Heidelberg (ln(537)); Ki-67 was detected with a mouse monoclonal IgG1 (NCL-L-Ki67-MM1; Novocastra) used at a dilution of 1∶100. Sections stained with k14.2 were pretreated by cooking for 20 minutes in citrate buffer, pH 6.0 and sections stained for Ki-67 were additionally pretreated with proteinase K for 15 minutes at 37°C. Staining was performed by streptavidin coupled to horseradish peroxidase or alkaline phosphatase, as described [60] (link).
9
Immunohistochemical Analysis of Endometriosis
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We performed immunohistochemical studies of target antigen in the serial section of biopsies derived from eutopic endometrium and from cyst walls of ovarian endometrioma using the following antibodies and concentrations: CD68 (marker of macrophages, 1:200), M0814, mouse monoclonal, Dako, Denmark; Ki-67 (cell proliferation marker, 1:100), NCL-L-Ki67-MM1, mouse monoclonal, Novocastra Laboratories Ltd, Newcastle, UK; CD31 (vascular cells marker, 1:50), M0823, mouse monoclonal, Dako, Denmark; COX2 (rate limiting enzyme for PG production, 1:100), CX-294, mouse monoclonal, Dako, Denmark; Fibronectin (cell adhesion marker, 1-400), ab2413, rabbit polyclonal, abcam, Tokyo, Japan. Non-immune immunoglobulin (Ig) G1 (1:50), a mouse monoclonal antibody from Dako, Denmark, was used as a negative control. A complete list of all antibodies, respective concentration used and positive controls is shown in Table 1.
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