Anti-mouse galectin-3/Mac2 (Rat IgG, 14–5301) was purchased from Bay Bioscience Co. Ltd (Hyogo, Japan). The deparaffinized sections were blocked for endogenous peroxidase activity by incubation in distilled water containing 3% hydrogen peroxide for 5 min. Antigen retrieval was performed using a 0.01 M citrate buffer (pH 6.0) for antigalectin-3 antibody by the Pascal heat-induced target retrieval system (Dako, Carpinteria, California, USA). Nonspecific binding sites were blocked in 0.01 M PBS, pH 7.4, containing 2% BSA (Wako Pure Chemical, Osaka, Japan) for 60 min. Antigalectin-3 antibodies used at a dilution of 1 : 100 in 2% BSA/PBS were added on the slides and incubated overnight at 4°C. Galectin-3 was detected using a biotinylated anti-rat IgG (1 : 200, E0468; Dako) for 30 min, followed by incubation with avidin-coupled peroxidase (Vectastain ABC kit; Vector Laboratories, Burlingame, California, USA) for 30 min. The peroxidase-binding sites were detected by staining with 3,3′-diaminobenzidine in 50 mM Tris-EDTA buffer, pH 7.6. Finally, counterstaining was performed using Mayer’s hematoxylin.
Anti rat igg
Manufactured by Agilent Technologies
Sourced in Denmark
Anti-rat IgG is a laboratory reagent used for the detection and quantification of rat immunoglobulin G (IgG) in various experimental and analytical applications. It serves as a specific binding agent for rat IgG, allowing for the identification and measurement of this antibody class.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate sample buffer, by Nacalai Tesque (2 mentions)
Polyacrylamide gel, by Nacalai Tesque (2 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Pascal heat induced target retrieval system, by Agilent Technologies (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine dab kit, by Vector Laboratories (1 mentions)
Nimp r14, by Abcam (1 mentions)
Lsab2 system hrp kit, by Agilent Technologies (1 mentions)
Peroxidase conjugated anti mouse igg, by Agilent Technologies (1 mentions)
1 step ultra tmb elisa, by Thermo Fisher Scientific (1 mentions)
Nunc maxisorp 96 well immunoplates, by Thermo Fisher Scientific (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Hrp conjugated anti mouse igg, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Aprotinin, by Nacalai Tesque (1 mentions)
Skim milk, by Nacalai Tesque (1 mentions)
Anti β actin, by Nacalai Tesque (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
6 protocols using anti rat igg
1
Immunohistochemical Detection of Galectin-3
2
Quantifying Inflammatory Cells in Nasal Tissues
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The heads of mice were fixed in 10% formalin, decalcified, and embedded in paraffin wax. Nasal tissues were sectioned and stained with hematoxylin and eosin (H&E) for inflammatory cell counting and with Sirius red for eosinophil counting. For neutrophils staining, sections were immunostained with the rat anti-mouse neutrophil antibody NIMP-R14 (Abcam, Cambridge, MA, USA) and subsequently with anti-rat IgG (Dako, Copenhagen, Denmark). Bound antibodies were visualized by 3.3’-diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA, USA).
3
Immunohistochemical Analysis of Myeloperoxidase
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Skin biopsies were fixed in 10% neutral buffered formalin, embedded in paraffin, routinely processed, sectioned at 5 μm, and stained with antibodies (Ab) recognizing myeloperoxidase (Dako). Detection of primary Ab binding was performed using biotinylated polyclonal rabbit anti-rat IgG, streptavidin horseradish peroxidase and AEC+ substrate (all from DAKO), or the LSAB2 System-HRP kit (DAKO); counterstaining was done with hematoxylin.
4
ELISA for hPDPN Binding Assessment
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Recombinant hPDPN or glycopeptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at a concentration of 1 μg/ml for 30 min. After blocking with 1% BSA in 0.05% Tween20/phosphate buffered saline (PBS, Nacalai Tesque, Inc.), the plates were incubated with culture supernatant followed by 1:1000 diluted peroxidase-conjugated anti-mouse IgG or anti-rat IgG (Dako; Agilent Technologies, Inc., Glostrup, Denmark). The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories Inc.).
5
Western Blot Analysis of EpCAM Expression
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Cell pellets were lysed in PBS with 1% Triton X-100 and 50 µg/ml aprotinin (cat. no. 03346-84; Nacalai Tesque, Inc.). Protein concentration was determined using the BCA assay. Cell lysates of CHO-K1, CHO/EpCAM, Caco-2 and BINDS-16 cells were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). The samples (10 µg/lane) were then electrophoresed on 5–20% polyacrylamide gels (Nacalai Tesque, Inc.) and transferred to polyvinylidene difluoride membranes (Merck KGaA). Following blocking with 4% milk (Nacalai Tesque, Inc.) for 1 h, the membrane was incubated with anti-EpCAM (1 µg/ml) or anti-β-actin (1 µg/ml; clone AC-15; cat no. A5441; Sigma-Aldrich; Merck KGaA) antibodies for 1 h, followed by incubation with HRP-conjugated anti-mouse IgG (cat. no. P0260, Agilent Technologies, Inc.) or anti-rat IgG (cat. no. A9542; Sigma-Aldrich; Merck KGaA) at a 1:2,000 dilution for 1 h at room temperature. The membrane was developed using the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) and a Sayaca-Imager (DRC Co., Ltd.). All western blotting procedures were performed at room temperature.
6
Immunoblotting Analysis of EpCAM Expression
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Cell pellets were resuspended in PBS with 1% Triton X-100 (cat. no. 168-11805; FUJIFILM Wako Pure Chemical Corporation) and 50 µg/ml aprotinin (product no. 03346-84; Nacalai Tesque, Inc.). Protein concentration was determined by BCA method. Cell lysates were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). The samples (10 µg/lane) were then electrophoresed on 5-20% polyacrylamide gels (Nacalai Tesque, Inc.) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 h, the membrane was incubated with an anti-EpCAM mAb (5 µg/ml) or anti-β-actin (1 µg/ml; clone AC-15; cat. no. A5441; Sigma-Aldrich; Merck KGaA) for 1 h, followed by incubation with HRP-conjugated anti-mouse immunoglobulins (cat. no. P0260; Agilent Technologies, Inc.) or anti-rat IgG (cat. no. A9542; Sigma-Aldrich; Merck KGaA) at a 1:2,000 dilution for 1 h. The membrane was developed using the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) and a Sayaca-Imager (DRC Co., Ltd.). All western blot procedures were performed at room temperature.
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