Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue using 4 μm sections cut from paraffin blocks. Staining was performed using a fully automated system (Benchmark ULTRA, Ventana Medical Systems, Tuscon, AZ, USA) and the following antibodies were used: NTRK1 (Ab76291, 1:1,500, ABCAM), SOX10 (383A-75,1:50, CELL MARQUE), S100 (Z0311, 1:8,000, DAKO), H3 (9733, 1:100, CELL SIGNALING), CD34 (790-2927, VENTANA), SMA (VPS281, 1:50, VECTOR), Desmin (760-2513, VENTANA), Melan A (790-2990, VENTANA), STAT6 (SC-621, 1:2,500, SANTA CRUZ) and HMB45 (M0634, 1:100, DAKO).
Hmb45
Manufactured by Agilent Technologies
Sourced in Denmark, United States, United Kingdom
The HMB45 is a laboratory equipment product from Agilent Technologies. It is designed for specific analytical tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information may be available from the company directly.
Automatically generated - may contain errors
Lab products found in correlation
Melan a, by Agilent Technologies (4 mentions)
Sox10, by Cell Marque (2 mentions)
Ae1 ae3, by Agilent Technologies (2 mentions)
Desmin, by Agilent Technologies (2 mentions)
S 100 protein, by Agilent Technologies (2 mentions)
Benchmark xt, by Roche (2 mentions)
Bond 3, by Leica (2 mentions)
Ab76291, by Abcam (1 mentions)
Desmin, by Roche (1 mentions)
Melan a, by Roche (1 mentions)
M0634, by Agilent Technologies (1 mentions)
Benchmark ultra, by Roche (1 mentions)
Clone 16, by Leica (1 mentions)
Bond max system, by Leica (1 mentions)
Cd20 l26, by Leica (1 mentions)
Zytochem plus hrp polymer kit, by Zytomed Systems (1 mentions)
Immpact novared, by Vector Laboratories (1 mentions)
Immpress ap reagent, by Vector Laboratories (1 mentions)
Tfe3 clone mrq 37, by Roche (1 mentions)
Sma clone 1a4, by Agilent Technologies (1 mentions)
31 protocols using hmb45
1
Comprehensive Immunohistochemical Profiling
2
Immunohistochemical Profiling of Tumors
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Immunohistochemical (IHC) staining was performed on representative 4-μm-thick formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks for the five cases using the Leica BOND-MAX™ system (Leica Biosystems, Wetzlar, Germany) according to the manufacturer’s protocol. We used antibodies for the ER (6F11, 1:300, Novocastra, New Castle, United Kingdom, 1:300), PR (clone 16, Novocastra, 1:1200), premelanosome protein (HMB-45, Dako, Carpinteria, CA, USA, 1:80), cytokeratin (PAN CK) (AE1/AE3, Dako, 1:500), CD 20 (L26, Novocastra, 1:800), CD 3 (polyclonal, Dako, 1:300), leukocyte common antigen (CD 45 RB) (2B11 + PD7/26, Dako, 1:1000) and CD 56 (CD564, Novocastra, 1:200).
3
Immunohistochemical Staining of Paraffin Sections
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Paraffin sections were incubated at 60 °C for 2 h and afterwards dehydrated with xylene and decreasing ethanol concentration, followed by distilled water. Antigen retrieval was performed using 10 mM sodium citrate solution (pH6) for 30 min in a water bath at 95 °C. The endogenous peroxidase activity of the sections was blocked by using 3% H2O2 for 10 min. Antigen detection, including blocking of background staining, was performed using a ZytoChem Plus HRP Polymer Kit (Zytomed Systems GmbH, Berlin, Germany). Tissue sections were incubated with primary antibodies for 30 min at 37 °C (Ki67 monoclonal antibody 1:10 (SP6, Thermo Fisher Scientific, Rockford, IL, USA ), MelanA monoclonal antibody 1:50 (A103, Abcam, Cambridge, UK), and antihuman melanosome 1:100 (HMB45, Dako, Carpinteria, CA, USA), followed by DAB/HRP detection. Sections were counterstained with Mayer’s hematoxylin solution for 30 s.
4
Immunohistochemistry for Melanoma Markers
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For immunohistochemistry, 5 μm tissue sections were steamed for 20 min with antigen retrieval solution and stained with primary antibodies, SOX10 (A-2, sc-365692; Santa Cruz Biotechnology, Santa Cruz, CA), HMB45 (M0634; Dako, Carpentaria, CA) and S100 (Z0311; Dako, Carpentaria, CA) overnight at 4 °C. On the next day, sections were incubated for 30 min in Imm-PRESS AP Reagent (Vector Laboratories, Burlingame, CA, USA), followed by 5 min incubation with ImmPACT NOVA-RED (Vector Laboratories).
5
Immunohistochemical Analysis of Sarcoma Markers
6
Western Blotting Antibody Validation
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Following antibodies were used for western blotting: Actin (I-19), Ezrin (C-19), Radixin (C-15), Moesin (C-15), Merlin (B-12), MYPT1 (H-130), c-myc (9E10) from Santa Cruz Biotechnology; CPI-17 (E305) from Epitomics; Ezrin (3C12) from Neomarkers; Ezrin (3145), Radixin (C4G7), phospho-ERM (3149) from Cell Signaling Technology; Moesin (EPR2429(2)) from Abcam; phospho-NF2 S518 from US Biologicals; VSVg from Roche. For Ras, either the antibody from the Active Ras Pulldown and Detection Kit from Thermo Fisher Scientific was used, or Ras (EP1125Y) from Abcam (Figure 4C ). For immunoblots of ERM (unless otherwise stated), a mixture of Ezrin (C-19), Radixin (C-15) and Moesin (C-15) antibody was used; for single Ezrin blots, the 3C12 antibody was used. Ezrin (3145), Radixin (C4G7) and Moesin (EPR2429(2)) were used for Figures 1D , 3A , 4A and Supplementary Figure S2 . Staining of paraffin sections was performed using anti-CPI-17 from Millipore and HMB45 from DAKO. Ras antibody Y13-259 (Thermo Fisher Scientific) was used for the GEF assay.
7
In Vivo Assessment and Histochemical Analysis of Transplanted Eyes
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In vivo assessment was performed by spectral domain optical coherence tomography (SD-OCT) (Envisu, Bioptigen) 1 week post transplantation surgery. Histological and immunohistochemical analysis was also performed. Enucleated eyes were cryopreserved according to previously published protocols (Cuenca et al., 2018 (link)). The tenon’s capsule and extraocular muscles were removed as much as possible, and a cut was made around the limbus with a scalpel blade to allow the fixative solution to enter into the eye cavity. Eyes were fixed in 4% paraformaldehyde for 2 h at room temperature. After washing with 0.1 M sodium phosphate buffer (pH 7.4), eyes were immersed in increasing concentrations of sucrose (up to 25%) before freezing. Cryosections (12 mm thickness) were collected and processed for immunofluorescent labeling with anti-Ku80 antibody (rabbit anti-human, 1:200, ab80592, Abcam), and melanosome antibody (mouse anti-human, 1:200, HMB45, Dako). DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear counterstaining (Molecular Probes). Fluorescence images were acquired with a confocal microscope (C2, Nikon).
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In vivo assessment was performed by spectral domain optical coherence tomography (SD-OCT) (Envisu, Bioptigen) 1 week post transplantation surgery. Histological and immunohistochemical analysis was also performed. Enucleated eyes were cryopreserved according to previously published protocols (Cuenca et al., 2018 (link)). The tenon’s capsule and extraocular muscles were removed as much as possible, and a cut was made around the limbus with a scalpel blade to allow the fixative solution to enter into the eye cavity. Eyes were fixed in 4% paraformaldehyde for 2 h at room temperature. After washing with 0.1 M sodium phosphate buffer (pH 7.4), eyes were immersed in increasing concentrations of sucrose (up to 25%) before freezing. Cryosections (12 mm thickness) were collected and processed for immunofluorescent labeling with anti-Ku80 antibody (rabbit anti-human, 1:200, ab80592, Abcam), and melanosome antibody (mouse anti-human, 1:200, HMB45, Dako). DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear counterstaining (Molecular Probes). Fluorescence images were acquired with a confocal microscope (C2, Nikon).
8
Comprehensive Immunohistochemical Profiling
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HE staining: All specimens were fixed by 10% neutral formalin, regularly dehydrated, paraffin-embedded, sectioned into 3-μm sections, and processed for HE staining. IHC staining: IHC staining was performed using the EnVision two-step strategy. Primary antibody information: CD117, HMB45, Desmin, synaptophysin, chromograninA, S100 protein, wide-spectrum cytokeratin, Ki67, and vimentin (DAKO); Bcl2, P53, CD56, CD57 (LEICA); muscle specific actin (MSA), CD34, H-caldesmon (LongIsland); SOX10, INI1, DOG-1 (Gene Tech); somatostatin receptor 2 (SSTR2), somatostatin receptor 5 (SSTR5) (Abcam); α-smooth muscle actin (α-SMA) (Thermo); ATRX (Sigma); calponin, Collagen type IV (MXB Biotechnologies); BRAF V600E (ZSGB Biotechnology). The dilutions, clone and sources of these antibodies were listed in Table 1 .
9
Immunohistochemical Analysis of Sebaceous Glands
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A total of 171 biopsy samples of the facial skin and the scalp which were randomly selected from the pathologic archives of the Department of Dermatology, Kyungpook National University Hospital, were used for the study. H&E staining of all samples (blocks of paraffin-embedded tissues) were reviewed retrospectively, and 103 samples with SGs were identified. Among them, 54 slides were already stained with the antiserum to S-100 (Dako, Ely, UK), 7 with antiserum to human melanoma black-45 (HMB-45; Dako), 11 with Fontana-Masson (F-M) stain (Junsei Chemical Co., Ltd., Tokyo, Japan), 4 with antiserum to CD1a (Novocastra, Newcastle, UK), 7 with antiserum to c-kit (Dako, Glostrup, Denmark), 10 with antiserum to microphthalmia-associated transcription factor (MITF; Leica Biosystems, Newcastle, UK), and 10 with antiserum to tyrosinase (Thermo Scientific, Fremont, CA, USA). All tissue sections contained epidermal melanocytes and Langerhans cells, which served as internal positive controls (Fig. 1 ).
10
Comprehensive Immunohistochemical Evaluation
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The tissue samples were fixed with 10% neutral formalin and embedded in paraffin. Tissue sections of 4 ~ 5 μm thickness were prepared and stained with hematoxylin and eosin (H&E), and additional sections were stained with Congo red. Immunohistochemical staining was performed using Dako EnVision Peroxidase detection system on Roche Ventana Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ). The primary antibodies panel consisted of calcitonin (CT) (clone SP17, Ready-to-Use), chromogranin A (CgA) (SP12, Ready-to-Use), synaptophysin (Syn) (SY38, 1:100), CD56 (1B6, 1:50, Novocastra, Newcastle upon Tyne, UK), TTF-1 (SPT24, prediluted), Thyroglobulin (TG) (2H11 + 6E1), Ki-67 (MIB-1, 1:100), carcinoembryonic antigen (CEA) (12–140-10, Ready-to-Use), cytokeratin (AE1/AE3, Ready-to-Use), epithelial membrane antigen (EMA) (E29, 1:100), vimentin (V9, 1:100), desmin (D33, 1:160), alpha-smooth muscle actin (SMA) (lA4, 1:100, Dako), HMB45 (HMB-45, 1:50), S-100 protein (polyclonal,1:200), HBME-1 (HBME-1, Ready-to-Use), galectin-3 (9C4, Ready-to-Use), CK19 (A53-B/A2.26, Ready-to-Use), and CD99 (12E7, 1:50). Unless otherwise stated, all antibodies were mouse monoclonal and from DAKO Corporation (Dako North America, Inc., Carpinteria, CA, USA). Appropriate positive and negative controls were run in parallel.
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