Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue using the Dako Envision Plus detection system (Dako, Carpinteria, CA, USA) with controls. The antibodies used included MDM2 (clone SMP14, ready-to-use; Abcam, Cambridge, UK), CDK4 (clone EP180, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), S-100 protein (clone 4C49, 1:100; Abcam, Cambridge, UK), CD34 (clone QBEnd 10, 1:100; Abcam, Cambridge, UK), desmin (clone D33, 1:100; Dako, Carpinteria, CA, USA), smooth muscle actin (SMA) (clone 1A4, 1:100; Dako, Carpinteria, CA, USA), H-caldesmon (clone h-CD, 1:100; Dako, Carpinteria, CA, USA), and p53 (clone Do-7, ready-to-use; Dako, Carpinteria, CA, USA).
Smooth muscle actin clone 1a4
Manufactured by Agilent Technologies
Sourced in United States, Denmark
Smooth muscle actin (clone 1A4) is a monoclonal antibody that binds to alpha-smooth muscle actin, a cytoskeletal protein found in smooth muscle cells. It can be used for the identification and localization of smooth muscle cells in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Desmin clone d33, by Agilent Technologies (3 mentions)
Cd68 clone pg m1, by Agilent Technologies (3 mentions)
Ferritin, by Agilent Technologies (2 mentions)
Chemmate envision detection kit, by Agilent Technologies (2 mentions)
Envision plus detection system, by Agilent Technologies (1 mentions)
P53 clone do 7, by Agilent Technologies (1 mentions)
Mdm2 clone smp14, by Abcam (1 mentions)
H caldesmon clone h cd, by Agilent Technologies (1 mentions)
Cd34 clone qbend 10, by Leica (1 mentions)
Cd8 clone c8 144b, by Agilent Technologies (1 mentions)
Bond max, by Leica (1 mentions)
Benchmark ultra stainer, by Roche (1 mentions)
Ae1 ae3 pck 26, by Roche (1 mentions)
Cd34 qbend 10, by Agilent Technologies (1 mentions)
Thrombin receptor, by Merck Group (1 mentions)
Myogenin clone f5d, by Agilent Technologies (1 mentions)
Myod1 clone 5.8a, by Agilent Technologies (1 mentions)
Envision hrp, by Agilent Technologies (1 mentions)
Cd163, by Santa Cruz Biotechnology (1 mentions)
7 protocols using smooth muscle actin clone 1a4
1
Immunohistochemical Analysis of Tumor Markers
2
Immunohistochemical Analysis of Splenic Stroma
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Splenic stroma and red pulp vessels were immunohistochemically studied with primary antibodies against the following antigens: (i) CD34 (clone QBEnd/10, Leica; Newcastle, United Kingdom); (ii) CD8 (clone C8/144B, Dako; Glostrup, Denmark); (iii) Smooth Muscle Actin (SMA, clone 1A4, Dako); (vi) CD68 (clone PGM1, Dako). Antigen detection was performed using an automated immunostainer (Bond-maX; Leica, Newcastle Upon Tyne, UK), as previously described (15) . In SCD and HS cases, the number of SMA-positive stromal cells was assessed by comparison with control spleens and graded as increased, normal, or reduced.
3
Immunohistochemical Analysis of Tumor Markers
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Immunohistochemical studies for cytokeratins AE1/AE3 (AE1/AE3/PCK26, Roche Diagnostics), CAM5.2 (Roche Diagnostics), and 5/6 (D5/16B4, Roche Diagnostics), as well as for p53 (DO-7, Roche Diagnostics), p63 (SSI6, 1:100; DCS), p40 (ΔNp63, polyclonal, 1:100; Zytomed), smooth muscle actin (clone 1A4, 1:400; Dako), desmin (DE-R-11, Roche Diagnostics), CD31 (JC70, Roche Diagnostics), CD34 (QBEnd/10, 1:200; Dako) and ALK1 (D5F3, 1:100; Cell Signaling) were performed on formalin-fixed paraffin embedded tumor tissue sections with BenchMark® Ultra stainer (Ventana, Tucson, AZ, USA).
Expression of p53 was recorded either as wild-type when heterogeneous nuclear staining was observed, or aberrant, including overexpression (neoplastic cells with uniformly strong nuclear staining indicating missense mutation) and complete lack of expression (null-phenotype indicating nonsense mutation) [8 (link)].
Expression of p53 was recorded either as wild-type when heterogeneous nuclear staining was observed, or aberrant, including overexpression (neoplastic cells with uniformly strong nuclear staining indicating missense mutation) and complete lack of expression (null-phenotype indicating nonsense mutation) [8 (link)].
4
Immunohistochemical Analysis of Carotid Arteries
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Paraffin-embedded carotid arteries were de-paraffinized in xylene, rehydrated in graded ethanol, and subjected to immunostaining. Immunohistochemistry was performed on serial sections, as described previously [12 (link)]. The primary antibodies used were CD74 (Sigma, St Louis, MO, USA), CD68 clone PG-M1 (Dako, Denmark), smooth muscle actin clone 1A4 (Dako, Denmark), ferritin (Dako, Denmark), and thrombin receptor (protease-activated receptor 1, Sigma). The immunoreactions were visualized using the EnVision+/horseradish peroxidase (Dako, Denmark) method and ChemMate EnVision Detection Kit (Dako, Denmark). Control sections without primary antibodies or with non-immune IgG were run for each protocol, resulting in consistently negative results. The slides were counterstained with haematoxylin.
All histological sections were examined under a light microscope, and the images were digitalized with Image Grabber program (Toronto, ON, Canada). The microscope was set on the same parameters used to scan all samples. The randomly digitalized images were analyzed with Adobe Photoshop (v5.5, Adobe Systems Incorporated, San Jose, CA, USA) as described previously [12 (link)]. The individual responsible for analysis was blinded to patient information.
All histological sections were examined under a light microscope, and the images were digitalized with Image Grabber program (Toronto, ON, Canada). The microscope was set on the same parameters used to scan all samples. The randomly digitalized images were analyzed with Adobe Photoshop (v5.5, Adobe Systems Incorporated, San Jose, CA, USA) as described previously [12 (link)]. The individual responsible for analysis was blinded to patient information.
5
Isolation of Tumor and Normal Gland Cells
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Crypts were isolated from tumor and normal tissues, as described previously, to obtain pure tumor glands separately from the surrounding stromal tissues (23 (link)–25 (link)). Tumor glands were isolated from the solid tumor region involved in the invasion front, and this involvement was confirmed using tissue sections prepared for the pathological diagnosis. Gland cells were obtained from the tumor tissues after careful separation of the stromal cells (i.e. CAFs) adjacent to the glands, performed under a dissecting microscope. Cells from normal fallopian tube tissue and normal fibroblasts within the Fallopian tubes were also obtained as controls. Paraffin-embedded tissue sections of the isolated samples were routinely processed to confirm the histology. Immunostaining using antibodies against smooth muscle actin (clone 1A4; Dako) and desmin (clone D33; Dako) was performed in the stromal cells to confirm the exclusive presence of fibroblasts, determined according to negative staining of smooth muscle actin and positive staining of desmin. However, we could not exclude the possibility of stromal cell contamination with other non-epithelial cells, such as inflammatory and vessel cells. Representative images are shown in Fig. 1 .
6
Histopathological and Immunohistochemical Analysis
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The surgical specimen was fixed in formalin and embedded routinely for histopathological evaluation. Hematoxylin and eosin (H&E) stained slides were prepared for histopathological examination and immunohistochemical staining was performed on 1 µm thick sections using an automated platform (Benchmark Ultra, Ventana Medical Systems Inc., Tucson, Arizona, U.S.A.) and the following antibodies: CD34 (clone QBEND-10; 1:50, Immunotech), smooth muscle actin (clone 1A4, 1:400, Dako), desmin (clone D33, 1:50, Dako), MyoD1 (clone 5.8A, 1:50, Dako), myogenin (clone F5D, 1:50, Dako), S100 (clone 4C4.9, 1:3000, Zytomed), MDM2 (clone IF2, 1:50, Calbiochem), CDK4 (clone DCS-156, 1:100, Zytomed), Retinoblastoma-1 (clone G3-245, 1:100, BD Pharmingen), CD10 (clone 56C6, 1:20, Zytomed), STAT6 (clone S-20, 1:1000, Santa Cruz) and SATB2 (clone EPNCIR130A, 1:200, abcam).
7
Carotid Artery Immunohistochemistry Protocol
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After surgical excision, carotid artery samples were collected and equally cut into 2 parts. One part was snap-frozen and saved at -80°C for biochemical analysis. Another part was dissected into 3 to 5 segments (≈5 mm), fixed in 4% formaldehyde and embedded in paraffin for serial sections.
Immunohistochemistry was performed on serial sections, as described previously. 11 (link) The primary antibodies used were CD68 clone PG-M1 (Dako, Denmark), smooth muscle actin clone 1A4 (Dako), CD86 (R&D Systems, United Kingdom), and CD163 (Santa Cruz Biotechnology, TX), ferritin (Dako), and TfR1 (Alpha Diagnostic International, TX). The immunoreactions were visualized using the EnVision+/HRP (horseradish peroxidase; Dako) method and ChemMate EnVision Detection Kit (Dako). Omission of the primary antibodies or isotype controls served as negative controls.
Immunohistochemistry was performed on serial sections, as described previously. 11 (link) The primary antibodies used were CD68 clone PG-M1 (Dako, Denmark), smooth muscle actin clone 1A4 (Dako), CD86 (R&D Systems, United Kingdom), and CD163 (Santa Cruz Biotechnology, TX), ferritin (Dako), and TfR1 (Alpha Diagnostic International, TX). The immunoreactions were visualized using the EnVision+/HRP (horseradish peroxidase; Dako) method and ChemMate EnVision Detection Kit (Dako). Omission of the primary antibodies or isotype controls served as negative controls.
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