Formalin-fixed samples were processed through paraffin embedment, sectioned at 5 μm (interscapular BAT, visceral (PGAT) and subcutaneous (SQAT) WAT) and stained in a 1:1200 dilution with UCP1 antibody (#U6382, 1:1000, Sigma-Aldrich; secondary antibody, #K400311-2, Envision Rabbit, Agilent) for 30 min with a heat-induced epitope retrieval (HIER) pretreatment, using DAKO brand citrate in a decloaking chamber. Sections were evaluated via an Olympus BX34 photomicroscope (Olympus, Melville, NY) and images were taken via an Olympus SC30 Optical Microscope Accessory CMOS color camera. Adipocyte size was calculated from three independent regions of the same 40× objective fields for SQAT, PGAT, and interscapular BAT depots (50 adipocytes/animal). Cross-sectional areas of the adipocytes were obtained from perimeter tracings using Image J software as previously described (Wainright et al., 2015 (link)). An investigator blinded to the groups performed all procedures.
En vision rabbit
Manufactured by Agilent Technologies
Sourced in France, Denmark
The Envision-Rabbit is a high-performance automated microplate reader developed by Agilent Technologies. It is designed to accurately measure absorbance, fluorescence, and luminescence in a wide range of microplate formats. The Envision-Rabbit offers fast and reliable data acquisition, making it a versatile tool for various applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Ucp 1 antibody, by Merck Group (1 mentions)
Sc30 optical microscope accessory cmos color camera, by Olympus (1 mentions)
Bx34 photomicroscope, by Olympus (1 mentions)
Panoramic midi 2, by 3DHISTECH (1 mentions)
Gill s hematoxylin 5, by Muto Pure Chemicals (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Hydrogen peroxidase, by Agilent Technologies (1 mentions)
2 methylbutane, by Merck Group (1 mentions)
Cck 8, by Merck Group (1 mentions)
Dm3000 led, by Leica (1 mentions)
Mec14, by Novus Biologicals (1 mentions)
Novared substrate, by Vector Laboratories (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Envision mouse, by Agilent Technologies (1 mentions)
Mac 3, by BD (1 mentions)
Interleukin 6 (il 6), by Santa Cruz Biotechnology (1 mentions)
P stat3, by Abcam (1 mentions)
7 protocols using en vision rabbit
1
Adipocyte Morphometric Analysis in BAT and WAT
2
Developmental Characterization of Mouse Gonad-Mesonephros
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The gonad-mesonephros complex was collected from the mouse fetus at E 12.5, 15.5, and 18.5 and embedded in para n, as previously reported (Omotehara et al. 2022b ). The para n blocks were serially cut at 5-µm-thick and placed sequentially on 10 slides, with 50-µm-interval sections per slide. The tissues from three males were analyzed at each developmental stage.
Immunohistochemistry was performed as in the previous report (Omotehara et al. 2022b ). The primary antibodies utilized are listed in Table 1. Details of the anti-AR rabbit antibody are described elsewhere (Prins et al. 1991; Vornberger et al. 1994) .
EnVision rabbit and EnVision mouse for DAB (Agilent Dako) were used as secondary antibodies for rabbit and mouse antibodies, respectively. The reacted antibodies were detected with 3-amino-9-ethylcarbazole (AEC substrate kit, Peroxidase (HRP), SK-4200, Vector), and Gill's hematoxylin V (Muto Pure Chemicals) was used for counterstain. The sections were digitized with a virtual slide scanner, Panoramic MIDI II (3DHISTECH), at ×20 magni cation. After the reacted antibodies were eliminated by autoclave at 121 degC for 20 min in 10 mM citrate buffer, pH6.0, the immunohistochemical procedures following the protein blocking were repeated to detect Collagen type IV in all sections.
Immunohistochemistry was performed as in the previous report (Omotehara et al. 2022b ). The primary antibodies utilized are listed in Table 1. Details of the anti-AR rabbit antibody are described elsewhere (Prins et al. 1991; Vornberger et al. 1994) .
EnVision rabbit and EnVision mouse for DAB (Agilent Dako) were used as secondary antibodies for rabbit and mouse antibodies, respectively. The reacted antibodies were detected with 3-amino-9-ethylcarbazole (AEC substrate kit, Peroxidase (HRP), SK-4200, Vector), and Gill's hematoxylin V (Muto Pure Chemicals) was used for counterstain. The sections were digitized with a virtual slide scanner, Panoramic MIDI II (3DHISTECH), at ×20 magni cation. After the reacted antibodies were eliminated by autoclave at 121 degC for 20 min in 10 mM citrate buffer, pH6.0, the immunohistochemical procedures following the protein blocking were repeated to detect Collagen type IV in all sections.
3
HMGB1 Immunohistochemistry in FFPE Tissue
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FFPE cancer tissue sections were deparaffinized in changes of xylène and rehydrated in absolute ethanol. Antigen retrieval was carried out by heating slides for 30 min in pH 6.0 citrate buffer at 98 °C. Endogenous peroxidase activity was inhibited by incubation with 3% hydrogen peroxidase (Dako, Trappes, France) for 15 min and then washed twice for 5 min with 0.025% Triton (v/v in TBS). Sections were then saturated 20min with Protein Block Serum Free (Dako, Trappes, France). Without washing, the primary antibody, a polyclonal rabbit anti HMGB1 antibody (ThermoScientist Pierce, PA1-16926) at the final concentration of 4μg/mL, was incubated overnight. After two washes in 0.025% Triton (v/v in TBS) sections were incubated by the secondary antibody (En vision-Rabbit, Dako, Trappes, France) for 45min, Upon two additional washes the peroxidase activity was revealed by means of daminobenzidine (DAB) substrate (Dako, Trappes, France), and the sections were counterstained with Mayer's hematoxylin.
4
Collagen IV Immunohistochemistry Post-MRI
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After MRI, few mice were used for immunohistochemistry: 12 h (n = 2), 24 h (n = 3), and 48 h (n = 2) after reperfusion. Animals were deeply anesthetized by intraperitoneal injection of ketamine/xylazine/acepromazine maleate (100/20/3 mg/kg body weight) and decapitated. Brains were immediately removed and snap-frozen in 2-methylbutane (Sigma-Aldrich, Switzerland) cooled with dry ice to − 30 °C and stored at − 80 °C until processing. They were then thawed and fixed in 4% paraformaldehyde (PFA) for 48 h, then trimmed (coronal section) and embedded routinely in paraffin wax. Consecutive sections (3–5 μm) were prepared and, after antigen retrieval via incubation in citrate buffer (pH 6.0) for 20 min at 98 °C, incubated, with rabbit anti-mouse collagen IV (Cat # 2150-1470, AbD Serotec, dil 1:200) for 15–18 h at 4 °C. Subsequently, they were incubated with Envision rabbit, Dako.
5
Immunohistochemical Visualization of CCK-8 Cells
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Formalin-fixed tissue samples were embedded in paraffin and 4-μm sections were cut. For paraffin-embedded tissue immunohistochemistry staining, deparaffinized and rehydrated sections were incubated with 3% hydrogen peroxidase solution for 10 min at room temperature and stained with primary antibodies against cholecystokinin-8 (CCK-8) (1:20,000, Sigma-Aldrich, St. Louis, MO, USA). The stained sections were further incubated with peroxidase-conjugated secondary antibodies (EnVision+Rabbit, Dako) for 30 min, and Mayer’s Hematoxylin was applied for counterstaining. The CCK-producing cells were observed using a light microscope (Leica, DM3000 LED, Wetzlar, Germany) attached to a camera (Dhyana 400DC, Tucsen Photonics, Fuzhou, China) with a capture program (Tucsen Mosaic version 2.2, Fuzhou, China).
6
Histological Analysis of Tumor Markers
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MCA205 and MC38 tumors were fixed in 4% formaldehyde using Sarstedt formalin system overnight at 4 °C. Samples were transferred to 70% ethanol and stored at 4 °C until paraffin embedding. Tissues were embedded in paraffin and cut into 4 μm tissue sections. Hematoxylin and eosin staining was performed according to standard protocols. For CD34 immunostaining, 10 mM citrate buffer, pH 6.0 antigen retrieval, and rat anti-mouse CD34 (1:400, MEC 14.7, Novus Biologicals) were used. For CD8 immunostaining, proteinase K buffer antigen retrieval, rat anti-mouse CD8a (1:50, 4SM16, Invitrogen) were used. For Ki67 immunostaining, Target Retrieval Solution, Citrate pH 6.1 (DAKO), rabbit anti-mouse Ki67 (1:100, SP6, Abcam) were used. The sections were then stained with polyclonal rabbit anti-rat IgG (1:200, Dako), or EnVision rabbit (Dako) with NOVAred substrate (Vector). The tissue sections were counterstained with hematoxylin. The percentage of CD8 + or Ki67+ cells were quantified with Qupath software [51 (link)], using positive cell detection with optimized parameters. The percentage of CD34 + areas were quantified with Qupath software (ver. 0.2.3), using trained positive pixel classifier.
7
Immunohistochemical Analysis of Aortic Inflammation
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Human (n ¼ 10 AAA samples and n ¼ 10 control atherosclerotic aorta samples) and murine tissue sections were deparaffinized and incubated overnight at room temperature with the primary antibody diluted in PBS, 1% BSA, using the following primary antibodies for the human studies: IL-6 (Santa Cruz Biotechnology, USA), IL-6R or CD126 (Abcam, UK) and pSTAT3 (Abcam, UK). Envision mouse or Envision Rabbit (Dako, Denmark) were used as secondary antibody [15] .
Murine sections were incubated with CD45 (BD Pharmingen, USA), MAC3 (BD Pharmingen,USA), MMP9 (Santa Cruz Biotechnology, USA) and Smooth Muscle Alpha Actin (DAKO, Denmark). Further sections were stained with Sirius Red for collagen and Weigert's elastin stain to visualize elastic lamina. Eight slides per animal were used per staining for analysis and only moderate or strongly reactive cells were counted as positive. The slides were blindly evaluated. A mean value for positive staining cells in the eight sections was calculated for each animal.
Murine sections were incubated with CD45 (BD Pharmingen, USA), MAC3 (BD Pharmingen,USA), MMP9 (Santa Cruz Biotechnology, USA) and Smooth Muscle Alpha Actin (DAKO, Denmark). Further sections were stained with Sirius Red for collagen and Weigert's elastin stain to visualize elastic lamina. Eight slides per animal were used per staining for analysis and only moderate or strongly reactive cells were counted as positive. The slides were blindly evaluated. A mean value for positive staining cells in the eight sections was calculated for each animal.
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