Immunohistochemical analyses were performed on representative tissue blocks using monoclonal antibodies to desmin (clone DE-R11, Dako), smooth muscle actin (SMA, clone 1A4, Dako), TFE3 (clone MRQ-37, Ventana), HMB45 (Dako), Melan-A (clone A103, LICR/in-house), pS6 (clone 91B2, CST) and pAKT(S473) (clone D9E, CST) for cases for which the original stained slides were not available for review. Immunohistochemistry was performed using a Leica Bond-3 automated platform (Leica, Buffalo Grove, IL), and a polymeric secondary kit (Refine, Leica) was used for the detection of the primary antibodies. Immunochemical results were evaluated by experienced histopathologists (RM and RAS) for the intensity of expression (weak, moderate or strong, compared to the intensity of staining of positive control tissue on the same slides) and percentage of tumor cells exhibiting expression. The results for pS6 were converted into an immunoreactive score (IRS), calculated as: intensity of staining (0 absent, 1 weak, 2 moderate, 3 strong) x percentage of cells staining (0–100), yielding IRS scores between 0 and 300. For the few cases for which tissue blocks or unstained slides for immunohistochemistry were not available, the immunohistochemical findings were obtained from the original pathology report issued by MSK gynecologic pathologists at the time of diagnosis.
Sma clone 1a4
Manufactured by Agilent Technologies
Sourced in United Kingdom, Denmark
The SMA clone 1A4 is a compact and versatile laboratory equipment designed for a variety of applications. It features a robust construction and is capable of performing essential tasks in a research or testing environment. The core function of this product is to provide reliable and consistent performance for users.
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Lab products found in correlation
Anti pcna antibody, by Abcam (2 mentions)
Bond 3, by Leica (1 mentions)
Hmb45, by Agilent Technologies (1 mentions)
Desmin, by Agilent Technologies (1 mentions)
Tfe3 clone mrq 37, by Roche (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Ab12188, by Abcam (1 mentions)
Anti mac3 clone m3 84, by BD (1 mentions)
Anti cd45 clone 30 f11, by BD (1 mentions)
Anti cd163 antibody, by Abcam (1 mentions)
Monoclonal mouse anti human cd68 clone pg m1, by Agilent Technologies (1 mentions)
Monoclonal mouse anti human cd3 clone f7.2.38, by Agilent Technologies (1 mentions)
5 protocols using sma clone 1a4
1
Immunohistochemical Analysis of Sarcoma Markers
2
Histological Analysis of Vascular Remodeling
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Paraffinized tissue sections (2.5 μm-thick) from experimental mice were stained with the following antibodies: anti-α-SMA (1:1000, SMA clone 1A4, DAKO), anti-PCNA (1:800, anti-PCNA antibody, Abcam) and anti-MMP-9 (1:400, MMP-9, Santa Cruz Biotechnology, Inc.). In each staining group, isotype controls without the primary antibody were included and exhibited no staining. In addition, αSMA and PCNA quantification was performed by counting positively stained cells in the intima and was expressed as the number of positive cells/high power field (HFP) in the intima. For the quantification of MMP-9, histological software (NIS-Elements AR ver5. 11.00) was used to measure the intimal MMP-9-positive area (μm2)/intima.
3
Immunohistochemical Analysis of Vascular Inflammation
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To detect the presence of inflammatory cells, vessel wall characteristics and effects of garcinol therapy, IHC was performed on paraffin-embedded sections of cuffs harvested after 3 days (ApoE*3-Leiden) or 21 days respectively (PCAF KO and WT mice). Weigert’s elastin staining was used to visualize elastic laminae. Inflammatory cell presence in the vascular wall was visualized using antibodies against leukocytes (anti-CD45 clone 30-F11, BD Pharmingen) and macrophages (anti-Mac3 clone M3/84, BD Pharmingen). Vascular SMCs were stained using anti-smooth muscle actin (SMA, clone 1A4, Dako) antibodies, PCAF was stained using anti-PCAF (ab12188, Abcam) antibodies and CCL2 was stained using anti-CCL2 (clone M-18, Santa Cruz) antibodies.
4
Immunohistochemical Characterization of Murine LCWE-Induced Inflammation
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Paraffinized sections (2.5 μm-thick) from LCWE-injected mice were stained with the
following antibodies: anti-mouse CD3 (1:200, monoclonal mouse anti-human CD3 clone
F7.2.38, Dako, Santa Clara, CA, USA), anti-CD20 (1:400, L26 monoclonal mouse antibody,
Nichirei Bioscience, Inc., Tokyo, Japan), anti-CD68 (1:500, monoclonal mouse anti-human
CD68 clone PG-M1, Dako), anti-CD163 (1:2000, anti-CD163 antibody, Abcam, Cambridge, UK),
α-smooth muscle actin (SMA, 1:1000, SMA clone 1A4, Dako), and anti-PCNA (1:800, anti-PCNA
antibody, Abcam). In each experiment, negative controls without the primary antibody were
included and showed no staining.
following antibodies: anti-mouse CD3 (1:200, monoclonal mouse anti-human CD3 clone
F7.2.38, Dako, Santa Clara, CA, USA), anti-CD20 (1:400, L26 monoclonal mouse antibody,
Nichirei Bioscience, Inc., Tokyo, Japan), anti-CD68 (1:500, monoclonal mouse anti-human
CD68 clone PG-M1, Dako), anti-CD163 (1:2000, anti-CD163 antibody, Abcam, Cambridge, UK),
α-smooth muscle actin (SMA, 1:1000, SMA clone 1A4, Dako), and anti-PCNA (1:800, anti-PCNA
antibody, Abcam). In each experiment, negative controls without the primary antibody were
included and showed no staining.
5
Thrombus Histomorphological Evaluation
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Formalin-fixed aspirates were embedded in paraffin, cut in 5 μm sections and stained with Haematoxylin and Eosin (HE) and elastic van Gieson (EvG) stains, respectively, for conventional histomorphological evaluation. The adjacent sections were used for immunohistochemical staining with anti-α smooth muscle actin (SMA, clone 1A4, Dako, GLostrup, Denmark) and anti-CD31 for smooth muscle cells (SMC) and endothelial cells, respectively, to visualize the organization of thrombus.
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