For immunohistochemical staining, formalin-fixed, paraffin-embedded human OSCC tissue sections were used. The primary antibodies used were as follows: anti-NOTCH3, rabbit polyclonal, 1:200 (ab23426, Abcam, CA, USA); anti-SMA, mouse monoclonal, 1:100 (M0851, Dako, Glostrup, Sweden). Antigen retrieval was performed according to the manufacturer’s protocol. EnVision+ Dual Link (Dako) was used for secondary antibody, and coloration was conducted with diamino-benzidine substrate. For immunofluorescent double-staining, anti-NOTCH3, 1:200 (Abcam), SMA, 1:100 (mouse monoclonal, M0851, Dako or rabbit monoclonal, Clone SP171, Spring Bioscinece, CA, USA), CD34, 1:100 (mouse monoclonal, NCL-L-END, Leica Biosystems, Wetzlar, Germany) and Cytokeratin, 1:100 (mouse monoclonal, Clone AE1/AE3, M3515, Dako) were used as a primary antibody. For antigen retrieval, the sections were treated with Tris buffer (pH = 7.4) containing 0.1 mg/ml trypsin for 30 min at 37°C for CD34 antibody. Alexa Fluor 488 goat anti-rabbit IgG (A11008, Invitrogen, CA, USA) and Alexa Fluor 594 goat anti-mouse IgG (A11005, Invitrogen) were used as secondary antibody. DAPI was used for nuclear staining. The immunofluoroscent images were captured and analyzed using Axioskop2 plus microscope (Carl Zeiss, Jena, Germany).
Clone ae1 ae3
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The Clone AE1/AE3 is a laboratory equipment product offered by Agilent Technologies. It is a tool used for the identification and differentiation of epithelial cells. The product provides a reliable and standardized method for the detection of cytokeratins AE1 and AE3, which are commonly expressed in epithelial tissues.
Automatically generated - may contain errors
Lab products found in correlation
Ventana benchmark xt, by Roche (4 mentions)
Bond polymer refine detection, by Leica (2 mentions)
Clone mib 1, by Agilent Technologies (2 mentions)
Bond max, by Leica (2 mentions)
Abc elite, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Clone 6f11, by Leica (2 mentions)
Biotinylated anti igg secondary antibodies, by Vector Laboratories (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 plus microscope, by Zeiss (1 mentions)
M0851, by Agilent Technologies (1 mentions)
Ab23426, by Abcam (1 mentions)
Envision dual link, by Agilent Technologies (1 mentions)
Ncl l end, by Leica (1 mentions)
Axio vert a1, by Zeiss (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Clone 2b11 pd7 26, by Agilent Technologies (1 mentions)
Clone 1a4, by Agilent Technologies (1 mentions)
Ae1 ae3, by Thermo Fisher Scientific (1 mentions)
23 protocols using clone ae1 ae3
1
Immunohistochemical Profiling of OSCC
2
Immunohistochemical Analysis of Tumor Samples
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All tumor samples were collected from the archives of the Institute of Pathology, University of Cologne (Cologne, Germany). The samples were formalin fixed and paraffin embedded (FFPE) as part of routine diagnostic procedures. Clinicopathologic data were obtained from case records provided by the Institute of Pathology, University of Cologne. All tumors were clinically and pathologically identified as being the primary and only neoplastic lesion and classified according to World Health Organization guidelines. Briefly, 3-μm-thick sections of FFPE tumors were deparaffinized, and antigen retrieval was performed by boiling the section in citrate buffer at pH 6 for 20 minutes. Primary antibodies used were given as follows: YAP (1:100, #4912; Cell Signaling Technology, Danvers, MA), endothelin-2 (EDN2; 1:100, NBP1-87942; Novus Biologicals, Littleton, CO), SAV1 (1:100, clone 3B3; Abnova, Taipei, Taiwan), and cytokeratin (1:200, clone AE1/AE3; Dako, Glostrup, Denmark). Staining was performed following established routine procedures, and staining intensity was evaluated individually in a blinded fashion. Statistical analysis was performed using Fisher exact test on GraphPad's QuickCalcs platform (http://graphpad.com/quickcalcs/contingency1.cfm ). P < .05 was considered statistically significant.
3
Cell Morphology and Immunohistochemistry of MaS-3 Cells
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Growth pattern and cell morphology was determined in vitro using a Zeiss phase-contrast microscope AxiovertA1 (Zeiss, Jena, Germany). For immunohistochemical assays were performed on formalin fixed paraffin-embedded MaS-3 cells. Briefly, freshly trypsinized MaS-3 cells from a T75 flask were 3 times washed with DPBS (Gibco) and centrifuged at 300 g to a pellet. For cytoblock preparation, this pellet was fixed in formalin incorporated into agarose, and subsequently paraffin-embedded. For immunohistochemical staining, the following antibodies and concentrations were used: pan-cytokeratin (1:1000; clone AE1/AE3, cat # M3515, Dako), vimentin (1:400; clone SP20, cat # RM-9120-s, Thermo Fisher Scientific), actin (1:200; clone 1A4, cat # M0851, Dako), LCA/CD45 (1:700; clone 2B11 PD7/26, cat # M0701, Dako), GATA3 (1:100; clone L50 823, cat # 390 M 16, Medac). Detection was done using the EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse (cat # K5007, Dako). All stainings were validated by internal and/or external positive controls as well as negative control specimens.
4
Detecting and Isolating Circulating Tumor Cells
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The formalin-fixed paraffin-embedded WBC pellets were sectioned, and nucleated CTCs were detected using anti-human pan-cytokeratin (CK, Clone AE1/AE3, Dako, Carpinteria, CA, USA) as described before [9 (link)]. CTCs were identified as hematoxylin-positive pan-CK+ cells. Cells on the slides processed with AccuCyte were labeled by multicolor immunofluorescence (IF) using the Ventana Discovery automated slide stainer. Slides were stained with DAPI (to mark nuclei), anti-human CK (AE1/AE3, eBioscience; C11, Biolegend), anti-mouse CD45 (30F11, Biolegend) cells, and a cocktail of antibodies against human cell surface markers [EpCAM (9C4, Biolegend), EGFR (EP38Y, Abcam), and HER2 (24D2, Biolegend)]. Stained slides were imaged by the CyteFinder® multi-channel scanning fluorescence microscope [13 (link)]. CyteMapper® software analyzed the scans and identified candidate cells that were presented to the reviewer for confirmation of CTC identity. Two independent reviewers identified CTCs from the scans and inconsistencies were resolved by consensus. CTCs were identified as DAPI+, human pan-CK+ and mouse CD45- cells. Individual CTCs were retrieved from slide by the CytePicker® module, which is integrated into CyteFinder as described previously, and placed into PCR tubes [13 (link)].
5
Immunohistochemical Staining Protocols for Pathological Analysis
6
Immunohistochemistry and In Situ Hybridization Protocol
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All the tissue samples were fixed in formalin and embedded in paraffin. Full-thickness tissue sections (4 μm thick) were used for immunohistochemistry (IHC) and in situ hybridization (ISH) in all cases. IHC staining and ISH was performed according to standard techniques on a Ventana Benchmark® XT autostainer (Ventana Medical Systems, Tucson, AZ, USA). Appropriate controls were included. We used the antibodies CD3 (1:50, Clone LN10; Novocastra, Newcastle upon Tyne, UK), CD20 (1:100, Clone L26, Dako, Glostrup, Denmark), CD138 (prediluted, Clone B-A38, Nichirei Bioscience, Tokyo, Japan) and cytokeratin (1:100, Clone AE1+AE3, Dako, Glostrup, Denmark) to detect T-lymphocytes, B-lymphocytes, plasma cells and residual epithelium, respectively. We further performed κ- and λ-ISH (Ventana Medical Systems) in HIC specimens and non-IC cystitis specimens.
7
Comprehensive Immunohistochemical Analysis
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Chromogen-based IHC analysis was performed by using an automated staining system (BOND-MAX; Leica Biosystems, Vista, CA) with antibodies against the following: pancytokeratin (epithelial cell marker; clone AE1/AE3, dilution 1:300; Dako, Carpinteria, CA), PD-L1 (clone E1L3N, dilution 1:100; Cell Signaling Technology, Danvers, MA), CD8 (cytotoxic T-cell marker; clone C8/144B, dilution 1:20; Thermo Fisher Scientific, Waltham, MA), CD3 (T-cell lymphocyte marker; clone D7A6E, dilution 1:100; Dako), PD-1 (clone EPR4877-2, dilution 1:250; Abcam, Cambridge, MA), Foxp3 (regulatory T-cell marker; clone 206D, dilution 1:50; BioLegend, San Diego, CA), KI67 (proliferation marker; clone MIB-1, dilution 1:100; Agilent Technologies, Santa Clara, CA), and CD68 (macrophage marker; clone PG-M1, dilution 1:450; Dako). Expression of all cell markers was detected using a Novocastra Bond Polymer Refine Detection Kit (catalog #DS9800; Leica Biosystems) with a diaminobenzidine reaction to detect antibody labelling and hematoxylin counterstaining. To guarantee specificity and sensitivity of the different antibodies, several tests were done until we obtained a reproducible pattern and correct geographical distribution of the different antibodies in the control tissues (Supplementary Fig. 1 ).
8
Immunohistochemical and Immunofluorescence Analysis of Human Tissue
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Human tissues were fixed in 10% neutral‐buffered formalin before embedding in paraffin. Sections were subjected to antigen retrieval using pH9 antigen retrieval buffer (DAKO Cat# S2375) at 95°C for 20 min. For immunohistochemistry, sections were incubated with antibodies against ER (Novocastra, Clone 6F11), PR (Novocastra, Clone 16), HER2 (Clone SP3, Spring Bioscience), pan‐cytokeratin (Clone AE1/AE3, DAKO) at 4°C overnight, followed by biotinylated anti‐IgG secondary antibodies (Vector Labs). Signal detection was performed using ABC Elite (Vector Labs) for 30 min and 3,3′‐diaminobenzidine (DAKO) for 5 min at room temperature. For immunofluorescence, sections were incubated with primary antibodies against CX3CR1 (BioLegend, Cat# 824001; 1:50 dilution), CK19 (Abcam, Cat# ab195872; 1:500 dilution), CD8 (Invitrogen, Clone SP16, 1:500 dilution), CD68 (DAKO, Clone PG‐M1; 1:200 dilution), Ki67 (BD Pharmingen, Clone B56; 1:100 dilution), Ki67 (Abcam, Cat# ab15580; 1:200 dilution), K8/18 (DSHB, Clone Troma1; 1:400 dilution), rabbit monoclonal platelet‐derived growth factor receptor ß (Cell Signaling, Clone 28E1, Cat# 3169; 1:100 dilution), pan‐cytokeratin (DAKO, Clone AE1/AE3, 1:500 dilution), CD31 (DAKO, Cat# M0823, 1:50 dilution) at 4°C overnight, followed by incubation of fluorophore‐conjugated secondary antibodies and DAPI (Invitrogen, Cat# D1306; 1:500 dilution).
9
Comprehensive Turtle Parasite Identification
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Sediment of freshwater baths of turtles was used to collect and identify parasites. Parasite specimens were collected and preserved in ethanol 70% for species identification. Complete necropsy was performed on each deceased individual within 6 h postmortem. Multiple tissues, including skin, fat, skeletal muscle, thymus, thyroid, heart, lung, liver, esophagus, stomach, intestine, spleen, pancreas, kidney, gonad, adrenal gland, salt gland and brain, were collected and fixed in 10% neutral formalin. All the tissues were routinely processed for histological examination and stained with hematoxylin and eosin (H&E) staining. To analyze gut contents of in situ B. manatorum immunohistochemical staining was undertaken for cytokeratin detection (monoclonal anti-cytokeratin, Isotype IgG1 Kappa, Clone AE1/AE3, Dako) using the avidin-biotin-peroxidase complex (ABC) method as recommended by the manufacturer. Swabs for bacteriology were taken aseptically from the coelomic cavity and the liver, lung and brain sections were frozen at −80 °C. Turtles behavior was checked there times a day as well as during feeding and treatment times. Animals were weighted once a week during the whole duration of the head-starting program.
10
Immunohistochemical Profiling of Tissue Specimens
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The 4 μm-thick, FFPE slices were de-paraffinized and rehydrated using a xylene and alcohol solution. Immunostaining was performed using automated instruments (Ventana Benchmark XT (Ventana Medical Systems) and or Dako Omnis (Dako, Carpinteria, CA, USA)) [1 (link),18 (link),19 (link),20 (link),21 (link),22 (link),23 (link),24 (link),25 (link),26 (link)]. After antigen retrieval, the slices were incubated with primary antibodies including pan-cytokeratin (pan-CK; 1:600, clone AE1/AE3, Dako), CK7 (Dako, 1:100, clone OV-TL 12/30, Dako), CK20 (1:100, clone Ks20.8, Dako), caudal type homeobox 2 (CDX2; 1:400, clone EPR2764Y, Cell Marque, Rocklin, CA, USA), paired box 8 (PAX8; 1:50, polyclonal, Cell Marque), estrogen receptor (ER; 1:150, clone 6F11, Novocastra, Leica Biosystems, Newcastle Upon Tyne, UK), progesterone receptor (PR; 1:100, clone 16, Novocastra), p53 (1:300, clone DO-7, Novocastra), p16 (prediluted, clone E6H4, Ventana Medical Systems), p63 (1:50, clone 4A4, Dako), and GATA-binding protein 3 (GATA3; 1:150, clone L50-823, Cell Marque). After chromogenic visualization, the slices were counterstained with hematoxylin. Appropriate positive and negative controls were concurrently stained to validate the staining method [27 (link),28 (link),29 (link)]. Negative controls were prepared by substituting non-immune serum for primary antibodies, resulting in no detectable staining.
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