Adipocyte area reflects metabolic function; larger adipocytes are typically insulin resistant and metabolically abnormal. However, range of metabolic health has not been previously correlated with breast adipocyte area. Individual adipocytes (≥100 cells/subject) were traced by Image J, areas were recorded for four fields of view and histograms of cell size distribution were generated. ND subjects (n=11) were compared T2D subjects (n=28). To assess CLS-B frequency, 42 samples of hematoxylin and eosin stained breast adipose tissue from ND and T2D subjects were examined with blinding to metabolic status and the number of CLS/section for each subject enumerated. Adipocyte-associated CD68 was confirmed by mouse monoclonal anti-human CD68 (PG-M1, Dako), with biotinylated goat anti-mouse IgG (Dako) as secondary.
Biotinylated goat anti mouse igg
Manufactured by Agilent Technologies
Sourced in United Kingdom, Denmark, United States
Biotinylated goat anti-mouse IgG is a secondary antibody conjugated with biotin. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pg m1, by Agilent Technologies (2 mentions)
Biotinylated swine anti rabbit igg antibody, by Agilent Technologies (1 mentions)
Non immune rabbit igg, by Merck Group (1 mentions)
Vector blue substrate, by Vector Laboratories (1 mentions)
Multiscreen hts 96 well elispot plates, by Merck Group (1 mentions)
Hl 1 medium, by Lonza (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Streptavidin alkaline phosphatase, by Vector Laboratories (1 mentions)
L glutamine, by Merck Group (1 mentions)
Sterile pbs, by Merck Group (1 mentions)
70 μm cell strainer, by Corning (1 mentions)
Biotinylated goat anti rabbit igg, by Agilent Technologies (1 mentions)
Streptavidin horseradish peroxidase complex, by Agilent Technologies (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
6 protocols using biotinylated goat anti mouse igg
1
Breast Adipocyte Size and Metabolic Health
2
Immunohistochemical Analysis of Placental IGF1R and Proliferation
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Following exposure to inhibitors and/or IGF, placental tissue was fixed in 4 w/v % paraformaldehyde overnight, embedded in paraffin wax and sectioned (5 µM). Sections were boiled in 0.1 μM sodium citrate buffer to maximize antigen retrieval and then incubated with rabbit anti-IGF1Rβ (1:100; Biosource, UK), mouse anti-Ki67 (MIB-1 clone, 1:200; DakoCytomation Ltd, Cambridgeshire, UK), non-immune rabbit IgG (Sigma, UK) or non-immune mouse IgG (DakoCytomation Ltd, Cambridgeshire, UK) followed by biotinylated swine anti-rabbit IgG antibody or biotinylated goat anti-mouse IgG (1:200; DakoCytomation Ltd, Cambridgeshire, UK). Staining was visualized using the avidin-peroxidase method with haematoxylin counterstain as previously described (Forbes et al., 2008b (link)) and images were captured using a Leica microscope. Levels of cytotrophoblast proliferation were then determined as previously described (Forbes et al., 2008b (link)) and expressed as a percentage of total cytotrophoblast number. Comparisons between groups were made using the Kruskal–Wallis test followed by a Dunn's post hoc test. Data were considered significant at P < 0.05.
3
Breast Adipocyte Size and Metabolic Health
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Adipocyte area reflects metabolic function; larger adipocytes are typically insulin resistant and metabolically abnormal. However, range of metabolic health has not been previously correlated with breast adipocyte area. Individual adipocytes (≥100 cells/subject) were traced by Image J, areas were recorded for four fields of view and histograms of cell size distribution were generated. ND subjects (n=11) were compared T2D subjects (n=28). To assess CLS-B frequency, 42 samples of hematoxylin and eosin stained breast adipose tissue from ND and T2D subjects were examined with blinding to metabolic status and the number of CLS/section for each subject enumerated. Adipocyte-associated CD68 was confirmed by mouse monoclonal anti-human CD68 (PG-M1, Dako), with biotinylated goat anti-mouse IgG (Dako) as secondary.
4
ELISPOT Assay for Murine Lymphocytes
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MultiScreen®HTS 96-well ELISPOT plates (Merck Millipore, Darmstadt, Germany) were coated overnight with either 10 μg/ml MP4 (Alexion Pharmaceuticals, Inc.) or 15 μg/ml anti-mouse IgG (MabTech, Nacka Strand, Sweden), while coating with sterile PBS (Sigma-Aldrich) served as a negative control. Plates were blocked with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in sterile PBS at room temperature for 2 h. Inguinal lymph nodes were disintegrated mechanically and filtered through a 70 μm cell strainer (Corning Inc., Corning, NY, USA). Cells were washed twice with complete RPMI-1640 medium (Gibco), subsequently resuspended in HL-1 medium (Lonza, Basel, Switzerland) containing 1% L-glutamine (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich) and plated at 1 × 106 cells/well followed by incubation at 37 °C and 7% CO2 for 24 h. Biotinylated goat anti-mouse IgG (Dako, Glostrop, Denmark) served as secondary antibody at 1:2000 dilution in 0.5% FBS/PBS + 0.025% Tween at 4 °C overnight. Plates were washed and incubated with streptavidin-alkaline phosphatase (Vector) at 1:800 dilution in 0.5% FBS/PBS for 2 h. Vector Blue substrate (Vector) was used for development. The spots were counted on an ImmunoSpot Series 6 UV Analyzer (CTL-Europe, Bonn, Germany).
5
Insulin Cell Apoptosis Quantification
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The apoptosis rate of insulin cells was determined by double immunocytochemical labelling for active Caspase-3 and insulin.
To determine insulin-positive cells, the biotinylated-streptavidin-peroxidase immunocytochemical method was carried out. For this, sections were incubated with monoclonal mouse anti-insulin (diluted 1:1000 in TBS, Sigma) overnight at 4°C, followed by incubation with biotinylated goat anti-mouse IgG (Dako, diluted 1:100, in TBS) and streptavidin-horseradish peroxidase complex (Dako, diluted 1: 100 in TBS), applied successively at room temperature for 40 min each. The reactions were developed in freshly prepared 3–3'DAB (0.025% in TRIS buffer containing 0.03% of H202). The second reaction for active caspase-3 was visualized by immunofluorescence. The sections were incubated with polyclonal rabbit anti-active caspase-3 (Sigma, diluted 1: 500 in TBS) overnight at 4°C, followed by incubation with biotinylated goat anti-rabbit IgG (Dako, diluted 1:100, in TBS) and Streptavidin-Cy3 complex (Sigma, diluted 1: 100 in TBS) applied successively at room temperature for 40 and 60 min, respectively.
To determine insulin-positive cells, the biotinylated-streptavidin-peroxidase immunocytochemical method was carried out. For this, sections were incubated with monoclonal mouse anti-insulin (diluted 1:1000 in TBS, Sigma) overnight at 4°C, followed by incubation with biotinylated goat anti-mouse IgG (Dako, diluted 1:100, in TBS) and streptavidin-horseradish peroxidase complex (Dako, diluted 1: 100 in TBS), applied successively at room temperature for 40 min each. The reactions were developed in freshly prepared 3–3'DAB (0.025% in TRIS buffer containing 0.03% of H202). The second reaction for active caspase-3 was visualized by immunofluorescence. The sections were incubated with polyclonal rabbit anti-active caspase-3 (Sigma, diluted 1: 500 in TBS) overnight at 4°C, followed by incubation with biotinylated goat anti-rabbit IgG (Dako, diluted 1:100, in TBS) and Streptavidin-Cy3 complex (Sigma, diluted 1: 100 in TBS) applied successively at room temperature for 40 and 60 min, respectively.
6
Oxidative Stress Quantification in Retina
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ROS levels were measured using an established method. 24 Eyes were embedded in OCT and immersed in liquid nitrogen and 10 lm cryosections stained with dihydroethidium (DHE, 5 lM in PBS; Sigma-Aldrich Corp.) for 30 minutes at room temperature. Four randomly selected sections per eye were selected randomly and labeling intensity was measured in the entire retina using ImageJ software. In addition, immunohistochemistry was performed for 8-hydroxy-2-deoxyguanosine (8-OHdG), an oxidized derivative of deoxyguanosine. Three lm paraffin sections of eyes were incubated with anti-8-OHdG (1:1000, #12501; QED Bioscience, San Diego, CA, USA) overnight at 48C. The sections then were washed with PBS and incubated for 45 minutes with biotinylated goat anti-mouse IgG (1:500, #E43301; Dako, Carpinteria, CA, USA), washed with PBS, and then incubated with the avidin-biotin complex (Vectastain ABC kit, #PK6100; Vector Laboratories, Burlingame, CA, USA). Labeling was developed with the 3,3 0 -diaminobenzidine substrate chromagen system (DakoCytomation) and the sections were coverslipped.
Immunolabeling for 8-OHdG was quantitated as described above for GFAP. Five rats per group were evaluated.
Immunolabeling for 8-OHdG was quantitated as described above for GFAP. Five rats per group were evaluated.
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