We analysed formalin-fixed paraffin-embedded whole coronal sections at levels 6–8 [27 (link), 42 ] containing the frontal cortex (Brodmann area 9). We ensured to select the cortical regions without any obvious infarction. Unless otherwise stated, 2–5 adjacent or alternate whole or half-coronal sections were used for the morphological analyses. Immunohistochemistry was performed to examine different microvascular structures essentially as described before [13 (link), 22 (link)]. The following antibodies were used to assess various cellular features: collagen IV (COL4 at dilation 1:1000, C1926, Merck (Sigma-Aldrich), Branchburg, NJ, USA), a marker of basement membrane in the vessels, platelet-derived growth factor receptor-β (PDGFR-β at 1:200 dilution, clone 42G12, #AF385, R&D systems, Minneapolis, MN, USA), a marker for pericytes, bone morphogenetic protein 4 (BMP4 dilution at 1:100, MBA1049, Millipore, MA, USA), α-smooth muscle actin (αSMA at dilution 1:1000, Clone 1A4, Dako, Cambridge, UK), a marker for mural cells, and glucose transporter-1 (GLUT-1 at 1:200, PA1-21,041, Fisher Scientific, Waltham, MA, USA), a marker of endothelial cells. Vectastain ABC mouse kits (PK-6102, Vector Laboratories, Burlingame, CA, USA) and Diaminobenzidine were used for single or double immunohistochemistry. Haematoxylin counterstain was used for ease in localising regions of interest.
Clone 1a4
Manufactured by Agilent Technologies
Sourced in Denmark, United Kingdom, United States
The Clone 1A4 is a lab equipment product from Agilent Technologies. It is a device designed for use in scientific research and laboratory settings. The core function of the Clone 1A4 is to perform specific tasks related to the analysis and manipulation of biological samples. No further details on the intended use or capabilities of the product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Mba1049, by Merck Group (2 mentions)
Pa1 21 041, by Thermo Fisher Scientific (2 mentions)
Vectastain abc mouse kits, by Vector Laboratories (2 mentions)
C1926, by Merck Group (2 mentions)
Af385, by R&D Systems (2 mentions)
Glut1, by Thermo Fisher Scientific (2 mentions)
A11058, by Thermo Fisher Scientific (2 mentions)
A11059, by Thermo Fisher Scientific (2 mentions)
Spinning disk invert, by Zeiss (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
α sma, by Agilent Technologies (2 mentions)
Clone v9, by Agilent Technologies (2 mentions)
Clone 6f2, by Agilent Technologies (2 mentions)
Clone gm008, by Agilent Technologies (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Clone ae1 ae3, by Agilent Technologies (1 mentions)
Clone 2b11 pd7 26, by Agilent Technologies (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
30 protocols using clone 1a4
1
Microvascular Analysis of Frontal Cortex
2
Immunohistochemical Analysis of Smooth Muscle Actin
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Immunohistochemical examinations of paraffin-embedded sections were performed using the avidin-biotin-peroxidase complex (ABC) method (VECTASTAIN Elite ABC
kit; Vector Laboratories, Burlingame, CA, U.S.A.). The primary antibody was a mouse monoclonal antibody against human alpha-smooth muscle actin (SMA) (clone
1A4, 1 in 100 dilution; Dako, Glostrup, Denmark). To remove endogenous peroxidase, the sections were immersed in 0.5% periodic acid solution at room temperature
for 20 min. The sections were then incubated in primary antibody solution at 4°C for 14 hr. After being washed with phosphate-buffered saline, the sections were
incubated in secondary antibody solution at room temperature for 30 min. After being incubated, the sections were reacted with ABC solution at room temperature
for 30 min. Visualization was accomplished using 3′3-diaminobenzedine solution. The sections were counterstained with Mayer’s hematoxylin. Sections without the
primary antibody were subjected to the same procedures as a negative control.
kit; Vector Laboratories, Burlingame, CA, U.S.A.). The primary antibody was a mouse monoclonal antibody against human alpha-smooth muscle actin (SMA) (clone
1A4, 1 in 100 dilution; Dako, Glostrup, Denmark). To remove endogenous peroxidase, the sections were immersed in 0.5% periodic acid solution at room temperature
for 20 min. The sections were then incubated in primary antibody solution at 4°C for 14 hr. After being washed with phosphate-buffered saline, the sections were
incubated in secondary antibody solution at room temperature for 30 min. After being incubated, the sections were reacted with ABC solution at room temperature
for 30 min. Visualization was accomplished using 3′3-diaminobenzedine solution. The sections were counterstained with Mayer’s hematoxylin. Sections without the
primary antibody were subjected to the same procedures as a negative control.
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
Immunofluorescence Staining of Tissue Sections
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Tissue sections were first treated with 0.1 mg/ml protease and then incubated overnight at 4 °C with primary antibodies to anti-COL4 (C1926 Sigma) monoclonal antibody, anti-PDGFR-β, (1:200 dilution, AF385, R&D Systems), αSMA (1:500 dilution, Clone 1A4, Dako), glucose transporter-1 (GLUT-1, 1:200, Thermo Scientific). Sections were washed with PBS and further incubated with donkey anti-goat conjugated Alexa Fluor 594 (1:1000, A11058, Thermo Fisher Scientific, Waltham, MA, USA) and rabbit anti-mouse Alexa Fluor 488 (1:1000, A11059, Thermo Fisher Scientific). Sections were then washed in PBS before mounting in Vectashield with DAPI (H-1200, Vector Laboratories). Images were captured using a Leica TCS SP2 (upright) and Zeiss Spinning Disk (Invert) confocal microscopes as described previously [12 (link)].
5
Analyzing Microvascular Changes in Frontal Lobe
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Formalin‐fixed paraffin‐embedded whole coronal sections at levels 6–8 (36 , 48 ) containing the frontal lobe (Brodmann area 9) were analyzed. When sampling tissue, we ensured to select the WM regions free of any obvious infarcts and thus assessed effects of remote stroke injury. Immunohistochemistry was performed to examine various microvascular structures essentially as described before (16 , 29 ). The following antibodies were used to assess various cellular features: collagen IV (COL4 at dilution 1:1000, C1926, Merck (Sigma‐Aldrich), Branchburg, NJ, USA), a marker of basement membrane in the vessels, platelet‐derived growth factor receptor‐β (PDGFR‐β at 1:200 dilution, clone 42G12, #AF385, R&D systems, Minneapolis, MN, USA), a marker for pericytes, bone morphogenetic protein 4 (BMP4 dilution at 1:100, MBA1049, Millipore, MA, USA), α‐smooth muscle actin (αSMA at dilution 1:1000, Clone 1A4, Dako, Cambridge, UK), a marker for mural cells and glucose transporter‐1 (GLUT‐1 at 1:200, PA1‐21041, Fisher Scientific, Waltham, MA, USA), a marker of endothelial cells. Vectastain ABC mouse kits (PK‐6102, Vector Laboratories, Burlingame, CA, USA) and DAB were used to localize single or double immunohistochemical stains. Tissue sections were then counter stained with hematoxylin to visualize the landmarks.
6
TGF-β1 and Indomethacin Effects on 3T12 Mouse Fibroblasts
7
Immunohistochemical Analysis of Ki-67 in Tumor Sections
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Tumor sections (5 μm) were blocked with 5% BSA in PBS and incubated with primary antibodies against Ki-67 (mouse anti-human Ki-67; 1 : 200, clone 1A4, Dako, Gostrup, Denmark). This step was followed by the addition of alkaline phosphatase conjugated polymer Mach 2 (Biocare Medical, Concord, USA) and visualized by Fast Blue BB/Naphthol-AS-MX-Phosphate, resulting in a blue/purple-colored precipitate. Hematoxylin and eosin staining was routinely preformed. Briefly, sections were deparaffinized in xylene I, II and III, washed and stained in hematoxylin (Harris Hematoxylin, Sigma) followed by eosin staining. In the next step they were dehydrated by washing in ethanol and mounted with DPX mountant and coverslip. Sections were imaged using Olympus microscope IX81 connected to Leica 200 scientific camera.
8
Cardiac Tissue Histochemical Assessment
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Hearts were removed, quickly washed in phosphate-buffered saline (PBS) (Gibco, Life
Technologies, São Paulo, Brazil) and cryopreserved in liquid nitrogen. Serial
transverse sections (8 mm) were obtained from a Leica cryostat (model 1850). Slides
were stained with hematoxylin and eosin (H&E) and modified with Gomori's
trichrome for morphological assessment. Immunohistochemistry was performed with
anti-fast myosin antibody for immunofluorescence (Sigma, St. Louis, MO) at a dilution
of 1:400. Slides were then incubated with secondary biotin-labeled affinity-isolated
anti-rabbit and anti-mouse immunoglobulins (LSAB® Kit, Peroxidase; DAKO
Corp., Carpinteria, CA).
Antigenic recovery of monoclonal mouse anti-human actin (smooth muscle) clone 1A4,
monoclonal mouse anti-human von Willebrand factor, and monoclonal mouse anti-actin
(sarcomeric) clone alpha-Sr-1 (Dako Cytomation, Denmark) was performed by enzymatic
digestion and 0.1% trypsin diluted in PBS, pH 7.4, in a 37ºC oven for one hour.
H&E staining identified inflammatory responses and morphological characteristics.
Factor VIII identified endothelial cells and blood vessels, actin 1A4 identified
smooth muscle fibers and sarcomeric anti-actin indicated cardiomyocytes.
Technologies, São Paulo, Brazil) and cryopreserved in liquid nitrogen. Serial
transverse sections (8 mm) were obtained from a Leica cryostat (model 1850). Slides
were stained with hematoxylin and eosin (H&E) and modified with Gomori's
trichrome for morphological assessment. Immunohistochemistry was performed with
anti-fast myosin antibody for immunofluorescence (Sigma, St. Louis, MO) at a dilution
of 1:400. Slides were then incubated with secondary biotin-labeled affinity-isolated
anti-rabbit and anti-mouse immunoglobulins (LSAB® Kit, Peroxidase; DAKO
Corp., Carpinteria, CA).
Antigenic recovery of monoclonal mouse anti-human actin (smooth muscle) clone 1A4,
monoclonal mouse anti-human von Willebrand factor, and monoclonal mouse anti-actin
(sarcomeric) clone alpha-Sr-1 (Dako Cytomation, Denmark) was performed by enzymatic
digestion and 0.1% trypsin diluted in PBS, pH 7.4, in a 37ºC oven for one hour.
H&E staining identified inflammatory responses and morphological characteristics.
Factor VIII identified endothelial cells and blood vessels, actin 1A4 identified
smooth muscle fibers and sarcomeric anti-actin indicated cardiomyocytes.
9
Immunohistochemical Analysis of PDGFR-β, α-SMA, and CD34
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TMA sections were deparaffinised, rehydrated and washed in distilled H2O. After antigen retrieval in decloaking chamber (Biocare Medical) at 110 °C for 5 min in pH 10.0 retrieval buffer, (for PDGFR-β) or pH 9.0 (for α-SMA), sections were incubated with blocking solution for 30 min and then incubated overnight with antibodies against PDGFR-β (No. 3169 Cell Signaling Technology, Danvers, MA, USA at dilution 1 : 100) or α-SMA (Clone 1A4; Dako, Inc., Denmark at dilution 1 : 300). Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Rabbit IgG MP-5401 or ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature and developed with Vector Blue AP Substrate Kit (SK-5300, Vector Laboratories).
To destroy alkaline phosphatases activity, sections were subsequently heated in decloaking chamber at 95 °C for 5 min, in pH 9.0 buffer, and then incubated with blocking solution for 30 min. Primary antibody against CD34 (Clone JC70A) at a 1 : 100 dilution was added for overnight incubation at 4 °C. Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories) for 1 h at room temperature and developed with Vector Red AP Substrate Kit (SK-5100, Vector Laboratories).
To destroy alkaline phosphatases activity, sections were subsequently heated in decloaking chamber at 95 °C for 5 min, in pH 9.0 buffer, and then incubated with blocking solution for 30 min. Primary antibody against CD34 (Clone JC70A) at a 1 : 100 dilution was added for overnight incubation at 4 °C. Sections were then incubated with polymer system (ImmPRESS-AP Polymer Anti-Mouse IgG, MP-5402, Vector Laboratories) for 1 h at room temperature and developed with Vector Red AP Substrate Kit (SK-5100, Vector Laboratories).
10
Immunofluorescence Staining of Tissue Sections
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Tissue sections were first treated with 0.1 mg/mL protease and then, incubated overnight at 4°C with primary antibodies to anti‐COL4 (C1926 Sigma) monoclonal antibody, anti‐PDGFR‐β, (1:200 dilution, AF385, R&D Systems), αSMA (1:500 dilution, Clone 1A4, Dako) and glucose transporter‐1 (GLUT‐1, 1:200, Thermo Scientific). Sections were washed with PBS and further incubated with donkey anti‐goat conjugated Alexa Fluor 594 (1:1000, A11058, Thermo Fisher Scientific, Waltham, MA, USA) and rabbit anti‐mouse Alexa Fluor 488 (1:1000, A11059, Thermo Fisher Scientific). Sections were then washed in PBS before mounting in Vectashield with DAPI (H‐1200, Vector Laboratories). Images were captured using a Leica TCS SP2 (upright) and Zeiss Spinning Disk (Invert) confocal microscopes as described previously (15 ).
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