Deoxy Big CHAP (DBC), 2-mercaptoethanol (BME), phenylmethylsulphonyl fluoride (PMSF), Triton X-100, and anti-FLAG M2 antibody-conjugated agarose beads (A2220) were purchased from Sigma-Aldrich. 16% w/v paraformaldehyde (PFA), Tween 20, glutaraldehyde (2.5%) in sodium cacodylate buffer, and anti-HA magnetic beads (88836) were purchased from Thermo Fisher Scientific. The antibodies used were as follows: FLAG (F1365; Millipore Sigma-Aldrich), RTN4 (sc-271878; Santa Cruz Biotechnology), RTN3 (A302-860A; Bethyl Laboratories), B12 (16780–1-AP; Proteintech Group), Hsp90 (sc7947; Santa Cruz Biotechnology), dsRNA (J2; Jena Biosciences; 10010200), NSP1 (GTX135612; Genetex), Streptavidin (LS-C203628; LifeSpan BioSciences), HA (11867423001; Roche), FAM134B (21537-1-AP; Proteintech Group), BAP31 (MA3-002; Invitrogen), DNAJB12 (16780-1-AP; Proteintech Group), anti-Mouse IgG Alexa Fluor 488 (A-11029; Invitrogen), anti-Rabbit IgG (A4914; Sigma-Aldrich), anti-Mouse IgG (A4416; Sigma-Aldrich), anti-Rat IgG (A5795; Sigma-Aldrich), and ProLong Diamond Antifade Mountant with DAPI (P36962; Invitrogen).
Anti rat igg
Manufactured by Merck Group
Sourced in United States, Germany
Anti-rat IgG is a laboratory reagent used to detect and quantify rat immunoglobulin G (IgG) in various biological samples. It is a polyclonal antibody that specifically binds to rat IgG and can be used in techniques such as ELISA, Western blotting, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Polyacrylamide gel, by Fujifilm (5 mentions)
Anti mouse igg, by Merck Group (4 mentions)
Sds sample buffer, by Nacalai Tesque (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Peroxidase conjugated anti mouse immunoglobulin, by Agilent Technologies (3 mentions)
Anti β actin clone ac 15, by Merck Group (2 mentions)
Peroxidase conjugated anti mouse igg, by Agilent Technologies (2 mentions)
Anti pa16 tag nz 1, by Merck Group (2 mentions)
Anti β actin ac 15, by Merck Group (2 mentions)
Axiocam digital camera, by Zeiss (2 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Deoxy big chap, by Merck Group (1 mentions)
Hsp90, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 antibody conjugated agarose beads, by Merck Group (1 mentions)
Fam134b, by Proteintech (1 mentions)
Dnajb12, by Proteintech (1 mentions)
Anti ha magnetic beads, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
23 protocols using anti rat igg
1
Immunoprecipitation and Immunofluorescence Protocol
2
Immunohistochemistry and Confocal Microscopy Protocol
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The sample preparation for the immunohistochemical analysis with confocal microscopy was performed as previously described (Backes et al. 2018 ). Briefly, 5-mm-thick sections collected from top and bottom internodes were vacuum-impregnated in the fixative solution containing 1% (w/v) glutaraldehyde, 2% (w/v) paraformaldehyde, 1% (w/v) caffeine, and 0.2 M sodium phosphate buffer (pH 7.2). After fixing overnight at 4 °C, the samples were dehydrated in an ethanol series (v/v): 70% for 30 min, 70% for 60 min, 95% for 30 min, 95% for 60 min, and 100% for 30 min. The samples were then embedded in the resin containing 2% (v/v) PEG 400 and 0.4% (w/v) dimethacrylate ethylene glycol and then included. Cross sections of 10 μm thickness were obtained using a microtome (Leica) and placed on glass slides. The sections were incubated with diluted antibodies (10-fold dilution for LM14/20 and 5-fold dilution for INRA-RU1/2) for 1.5 h, washed three times with PBS, and then incubated for 1.5 h with 100-fold diluted secondary antibodies (Sigma; anti-rat IgG coupled to FITC for LM14/20 and anti-mouse IgG coupled to FITC for INRA-RU1/2). The sections were rinsed three times with PBS, mounted in 50% (v/v) glycerol, and analyzed by a Zeiss LSM 880 confocal microscopy (Carl Zeiss AG). The wavelength of excitation and emission was set at 488 and 602 nm, respectively.
3
Monoclonal Antibody Characterization of RG-I
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Anti-rat IgG and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The series of monoclonal antibodies used to assess the polymers present in the isolated RG-I fractions were kindly provided by Professor Paul Knox from the University of Leeds. Sephadex G-25, DEAE-Sepharose Fast Flow, and Sepharose CL-6B were purchased from GE Healthcare (Uppsala, Sweden). All other reagents and chemicals were commercially available, of analytical grade, and produced in China.
4
Depletion of Macrophages In Vivo
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To deplete macrophages in vivo, we used the anti–colony stimulating factor 1 (CSF‐1) receptor antibody (ASF98), a rat monoclonal anti‐murine CD115 antibody (immunoglobulin G [IgG] 2a) that inhibits CSF‐1–dependent cell growth by blocking the binding of CSF‐1 to its receptor (CSF‐1R), the selectivity of which has been characterized previously.13 As a control, we injected an isotype‐matched anti‐rat IgG (Sigma‐Aldrich). Mice were injected intraperitoneally at doses of 2 mg/mouse.
Clodronate, encapsulated in liposomes, was also used to deplete macrophages in Mdr2−/− and CD1‐nude mice. Clodronate liposomes were prepared as described previously.14 Control liposomes contained phosphate‐buffered saline only. Each animal received 0.01 mL/g (5 mg of clodronate per 1 mL of the total suspension volume) of clodronate liposomes or control liposomes via intraperitoneal injection. The clodronate and control liposomes were obtained from the Foundation Clodronate Liposomes (Amsterdam, Netherlands).
The murine macrophage cell line RAW264.7 (ECACC) was grown in DMEM (Lonza) supplemented with 10% FBS NA (HyClone), 1% glutamine (Euroclone) and 1% penicillin/streptomycin (Life Technologies) under 5% CO2 at 37°C. Normal bone marrow macrophages were derived from of C57/BL6 mice (Harlan).15
Clodronate, encapsulated in liposomes, was also used to deplete macrophages in Mdr2−/− and CD1‐nude mice. Clodronate liposomes were prepared as described previously.
The murine macrophage cell line RAW264.7 (ECACC) was grown in DMEM (Lonza) supplemented with 10% FBS NA (HyClone), 1% glutamine (Euroclone) and 1% penicillin/streptomycin (Life Technologies) under 5% CO2 at 37°C. Normal bone marrow macrophages were derived from of C57/BL6 mice (Harlan).
5
Immunohistochemical Staining of CD45 in Tissue Sections
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The fresh frozen sections incubated with the rat-anti-CD45 IgG were processed as described above. After incubation with rat-anti-CD45 (1:50), the sections were rinsed 3 × 15 minutes in TBS, incubated with alkaline phosphatase conjugated anti-rat IgG (Sigma A8438) diluted 1:50, rinsed 3 × 15 minutes in TBS, followed by a 15 minute rinse in 10 mM Tris-HCl buffer (pH 9.5), and development in a Tris-HCl-MgCl2 buffer (pH 9.5) containing nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate yielding a bluish-black reaction product [27 (link)]. Sections were finally rinsed in TBS followed by H2O and coverslipped with Aquatex (Merck Millipore, Darmstadt, Germany).
6
Western Blot Analysis of BCRP Protein
7
Protein expression analysis of mouse hippocampus
8
Western Blot Analysis of Protein Samples
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Cell lysates were prepared by 1% Triton X-100 and cell debris was removed by centrifugation. Cell lysates were boiled in sodium dodecyl sulfate sample buffer with a reducing reagent (Nacalai Tesque, Inc.). These proteins (10 µg) were electrophoresed on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubated with 10 µg/ml of C20Mab-11, 1 µg/ml of NZ-1 (anti-PA tag) or 1 µg/ml of anti-β-actin for control (clone AC-15; Sigma-Aldrich Corp.), followed by incubation with secondary antibody peroxidase-conjugated anti-mouse immunoglobulin (1:1,000; Agilent Technologies Inc., Santa Clara, CA, USA) or anti-rat IgG (1:10,000; Sigma-Aldrich Corp.), respectively. Finally, proteins were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co. Ltd.).
9
Immunolocalization of Pectin Epitopes in Tomato
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The tomato pericarp was embedded into Steedman’s wax and cut to a thickness of 12 μm using a microtome (HM355 Microm) and collected on polylysine-coated slides. The slides were incubated in 97% (v/v) ethanol for 10 min three times and then rehydrated in 90% (v/v) ethanol for 10 min and in 50% (v/v) ethanol for another 10 min at room temperature (RT). Finally, the slides were washed in dH2O for 10 min. The water was changed, and the slides were washed for another 90 min.
Non-specific binding sites were blocked by incubation with 3% (w/v) milk protein in MP/PBS for at least 30 min. The slides were incubated with rat monoclonal antibody JIM5 that had been diluted fivefold in MP/PBS for at least 1 h at RT or overnight at 4°C and then washed three times with 1 × PBS with at least 5 min for each wash. The slides were incubated with a 100-fold MP/PBS-diluted secondary antibody [anti-rat-IgG (whole molecule) linked to FITC (Sigma)] for 1 h at RT. The slides were washed three times with PBS with at least 5 min for each wash. The slides were mounted using an antifade reagent Citifluor AF1 (Agar Scientific, Stansted, United Kingdom). Finally, these slides were examined and imaged using a fluorescence microscope (Leica Microsystems). Three biological repeats have been done to confirm this result.
Non-specific binding sites were blocked by incubation with 3% (w/v) milk protein in MP/PBS for at least 30 min. The slides were incubated with rat monoclonal antibody JIM5 that had been diluted fivefold in MP/PBS for at least 1 h at RT or overnight at 4°C and then washed three times with 1 × PBS with at least 5 min for each wash. The slides were incubated with a 100-fold MP/PBS-diluted secondary antibody [anti-rat-IgG (whole molecule) linked to FITC (Sigma)] for 1 h at RT. The slides were washed three times with PBS with at least 5 min for each wash. The slides were mounted using an antifade reagent Citifluor AF1 (Agar Scientific, Stansted, United Kingdom). Finally, these slides were examined and imaged using a fluorescence microscope (Leica Microsystems). Three biological repeats have been done to confirm this result.
10
Protein Extraction and Western Blot Analysis of Drosophila Embryos
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Total protein was extracted from 100 fly embryos using 50µl RIPA buffer (AuRAGENE, Cat. no. P002A) and 1µl cocktail (Biotool, Cat. no. B14001). For Western analysis, samples were mixed with 5 x loading buffer and boiled at 95°C for 10 min. Samples were then separated by 12% SDS-PAGE and transferred, membranes were blocked with 8% nonfat milk in TBST buffer (10 mMTris pH 7.4, 0.8% NaCl, 0.1% Tween-20). Primary antibodies were directed against the dNulp1 (1:2,000), and β-actin (1:2,000, Sigma). Secondary antibodies were anti-rat IgG (1:2,000; Sigma) or anti-mouse IgG (1:2,000; Sigma). Proteins were visualized using the Super Signal West Dura detection reagent (Thermo Scientific) and signals were detected with the ChemiGenius Bio Imaging System.
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