T cells (0.6×106) were lysed in (35 μl) Carin lysis buffer (20 mM Tris-HCl [pH 8.0], 138 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% Nonidet P-40, 10% glycerol, 2 mM Na vanadate) supplemented with complete protease inhibitors (Roche). Proteins were separated on 10% SDS-polyacrylamide gels and blotted onto PVDF membranes according to standard protocols. The following primary antibodies were used: anti-actin (AC-74; Sigma-Aldrich, St. Louis, MO), mouse anti-Runx3 (2B3; Abcam) and rabbit anti-Th-POK 22 (link). Secondary antibodies were a peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories) and a HRP-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch Laboratories). Signals were detected using ECL (SuperSignal West Dura Extended Duration Substrate from ThermoScientific).
Affinipure goat anti rabbit igg h l
Manufactured by Jackson ImmunoResearch
Sourced in United States
AffiniPure Goat Anti-Rabbit IgG (H+L) is a secondary antibody produced in goats and purified against rabbit immunoglobulins. It is designed to bind to the heavy and light chains of rabbit IgG antibodies, providing a means to detect and visualize rabbit primary antibodies in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Affinipure goat anti mouse igg h l, by Jackson ImmunoResearch (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Anti actin ac 74, by Merck Group (3 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Mouse anti runx3 2b3, by Abcam (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Anti th, by Merck Group (1 mentions)
Gtx635679, by GeneTex (1 mentions)
Anti ndufa9, by Proteintech (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Anti cox4, by GeneTex (1 mentions)
Anti cox2, by Proteintech (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx stain free precast gel, by Bio-Rad (1 mentions)
39 protocols using affinipure goat anti rabbit igg h l
1
Western Blot Analysis of T Cell Proteins
2
Immunohistochemical Labeling of Fos, ADA, Orexin, and TH
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Free-floating sections were incubated overnight at room temperature in the primary antibody (Fos polyclonal antibody, Ab-5 rabbit polyclonal, from Oncogene, San Diego, CA, diluted 1:20,000), incubated for 1 hour in the secondary antibody (Biotin-SP- conjugated AffiniPure goat anti-rabbit IgG H+L; from Jackson ImmunoResearch, PA, diluted 1:1,000), rinsed, and incubated for 1 hour in Vectastain ABC Elite Kit (Vector Laboratories, CA, diluted 1:500). Finally, the sections were revealed with 0.05% 3–3’diaminobenzidine hydrochloride (DAB) and nickel chloride, producing an enhanced dark blue reaction product. Selected sections already immunostained for nickel-enhanced Fos-ir were subjected to a second immunostaining to identify ADA-ir neurons in the TMN, or tyrosine hydroxylase (TH)-ir neurons in the VTA and locus coeruleus, or orexin-ir neurons in the LHA. The second immunostaining was revealed with DAB without nickel intensification, yielding a brown cytoplasmic precipitate that contrasted with the dark blue nuclear DAB-nickel labeling of the Fos-ir. The antisera used were anti ADA (polyclonal, raised in rabbit, diluted 1:5,000 from Chemicon, CA); anti-orexin A (rabbit polyclonal, 1:20,000; from Phoenix Pharmaceutical Inc., CA) and anti-TH (rabbit polyclonal, 1:10,000; from Chemicon, CA). Specificity of these antibodies was tested before [4 (link)].
3
Histopathological Analysis of SARS-CoV-2 Infection
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The lung (right upper lobe), brain (right hemisphere) and nasal turbinates were collected and placed in 10% formalin for >72 hrs. Fixed tissues were embedded in paraffin and tissue sections (3 μm) were processed for histology and immunohistochemistry (IHC). For histologic examination, slides were stained with hematoxylin & eosin (H&E). Scoring of lung histopathological features was based on18 (link),19 (link) and detailed in Table 3 . Quantitative morphometric analysis of lung pathology (% Lesion) was performed using two softwares: Interactive Learning and Segmentation Toolkit (Ilastik version 1.3.3) and CellProfiler Analyst software (version 3.1.5). Probability maps for the lesion area and whole lung are created in Ilastik and analysed in the CellProfiler Analyst software.71 (link) For viral detection, an IHC assay was performed using an anti-SARS-CoV-2 nucleocapsid protein rabbit IgG monoclonal antibody (GeneTex; GTX635679) at 1:1000 dilution and a peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, 111-035-144) at 1:500 dilution. Images were acquired with a NanoZoomer 2.0- HT Whole Slide Imager, Digital Pathology Slide Scanner.
4
Mitochondrial Protein Extraction and Western Blot
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Cells were lysed in RIPA lysis buffer (#R0278, Sigma) with the addition of the Halt Protease Inhibitor Cocktail (#8778, Thermo Scientific) and Halt Phosphatase Inhibitor Cocktail and EDTA solution (#78420, Thermo Scientific). The antibodies used included anti-NDUFA9 (#20312-1-AP, Proteintech), anti-COX4 (#GTX114330, GeneTex), anti-COX2 (#55070-1-AP, Proteintech), anti-β-actin (#MAB1501, Millipore), peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (#115-035-003, Jackson ImmunoResearch), and peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (#111-035-003, Jackson ImmunoResearch). The signals were developed using the Ultra ECL-HRP Substrate (#TU-ECL02, TOOLS) using X-ray films.
5
Cleaved Caspase-3 Expression in PART1-Depleted HCC1806 Cells
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HCC1806 cells with PART1 knockdown (through shRNA or GapmeR treatment) for 48h were lysed in RIPA buffer. 50 µg of the cytoplasmic lysate was loaded along with cleaved caspase-3 control cell extracts (#9664, Cell Signaling, New England Biolabs Ltd, Whitby, ON, Canada) in a Mini-PROTEAN TGX Stain-Free Precast Gel (Bio-Rad) and ran for 1 h at 100 V in Tris-Glycine-SDS buffer. The lysates were transferred onto PVDF membranes in a Transblot-Turbo Transfer system (Bio-Rad) and blocked in 5% BSA for 1h at room temperature. The membrane was incubated with cleaved caspase-3 (Asp175, 5A1E) rabbit monoclonal antibody (#9663, Cell Signaling, 1:1000 in 5% BSA) overnight at 4 °C followed by peroxidase affiniPure goat anti-rabbit IgG (H + L, #111-035-144, Jackson Immunoresearch, West Grove, PA, USA) antibody (1:1000 in 5% BSA) for 1 h at room temperature. The chemiluminescence was imaged with the ChemiDoc imaging system (Bio-Rad). Subsequently, the blot was re-probed for actin (#13E5, Cell Signaling) and similarly imaged.
6
Dot Immunoassay for Ara6 Glycoside Antibodies
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The specificity of the obtained antisera to both Ara6 glycosides 1 and 2 was tested by the dot immunoassay (titrated twice in the microtiter plate) as follows. An aliquot (1 μL) of solution of each antigen (1 or 2 ) in the double dilutions (initial concentration, 1 mg·mL−1) was spotted onto a Westran S polyvinylidene fluoride (PVDF) membrane (Whatman), and the membrane was incubated in a dry-air thermostat at 60 °C for 15 min. After spotting and drying, the membrane was then blocked for 12 h with 2% powdered milk diluted in 10 mM PBS. This procedure was performed to prevent a nonspecific antibody adsorption. Then the membrane was incubated in the obtained rabbit antisera, diluted 1:50 with 0.01 M PBS, at room temperature overnight. The membrane was washed four times at 15 min intervals with PBS containing 0.02% Tween 20. Then, it was incubated in a solution of peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories; diluted 1:2000) in PBS containing 0.02% Tween 20 and 0.02% powdered milk for 90 min. The membrane was washed four times for 15 min with PBS containing 0.02% Tween 20. After that, the membrane was treated with a substrate mixture of 0.05% 3,3′-diaminobenzidine and 0.02% hydrogen peroxide in 0.15 M PBS until intense brown dots appeared (Figure 5 ).
7
Immunofluorescence Staining of Murine Tissue
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For analysis of murine tissue samples, histology was performed using paraffin embedding. Tissue samples were fixed overnight in 4% formalin, dehydrated with ethanol, and subsequently embedded in paraffin. This was followed by immunofluorescence staining. Chamber slides and deparaffinized sections were blocked for 18 h at 4°C with PBS containing 10% FBS (Merck Millipore, Billerica, Waltham, MA, USA). Primary antibodies detecting NE (ab21595, Abcam, Cambridge, UK) or citH3 (ab5103, Abcam, Cambridge, UK) were added to the slides in a 1:200 dilution and incubated for 1.5 h at room temperature. This was followed by incubation with the Cy5-conjugated secondary detection antibody AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson Immuno Research Labs, West Grove, PA, USA) in a dilution 1:400 for 1 h at room temperature in the dark together with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). Slides were washed with PBS and H2O and samples were embedded in DAKO fluorescent mounting medium (Agilent Technologies, Santa Clara, CA, USA). Slides were analyzed either using the Eclipse Ni-U (Nikon Corporation, Tokyo, Japan) or the BZ-X700 microscope (Keyence Corporation, Osaka, Japan). Z-stacks were performed to increase depth of field. Post-processing of pictures was performed using Photoshop CS5 (Adobe, München, Germany).
8
Immunofluorescence Staining of Neuropilin-Expressing Macrophages
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Immunofluorescence double staining using paraffin-embedded tissue sections was performed as previously described [15 (link)]. IF was performed using antibodies against NRP-1 (rabbit polyclonal, Abcam, Cambridge, United Kingdom), NRP-2 (C-9, mouse monoclonal IgG2b, Santa Cruz Biotechnology, CA, USA), CD68 (PG-M1, mouse IgG1κ, DAKO, Glostrup, Denmark), CD163 (10D6, mouse IgG1, NOVOCASTRA, Newcastle, United Kingdom), HO-1 (D-8, mouse IgG1, Santa Cruz Biotechnology) and DC-SIGN (rabbit polyclonal, IgG, Abcam). In brief, following antigen retrieval, tissue sections were incubated overnight with a cocktail of primary antibodies, followed by fluorescein-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA) and rhodamine-conjugated AffiniPure donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories). Double IF was done to explore the co-expression of CD68, CD163 and HO-1 on NRP-1+ AMs, and co-expression of DC-SIGN on NRP-2+ AMs.
9
Western Blot Analysis of Seedling Proteins
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Seedlings grown under Rc irradiation for 9 days were harvested and weighed. Aerial parts of the seedlings were ground in a mortar and pestle in 25 mM HEPES-NaOH buffer (pH 7.5) containing 20 mM NaCl, 6 mM MgCl2 and 1 mM EDTA. Proteins in the extracts were mixed with same volume of 2× SDS-PAGE loading buffer. Resultant extracts from 0.67 mg of fresh weight of samples were subjected to SDS-PAGE using gels containing 15% (w/v) acrylamide and 6 M urea. Separated proteins in the gels were blotted onto a nitrocellulose membrane (Optitran BA-S 85 Reinforced NC, Schleicher & Schuell, http://www.gelifesciences.com/ ) electrophoretically. The procedures for immunochemical detection were carried out as described elsewhere [15 (link)] using alkaline phosphatase-conjugated AffiniPure Goat Anti Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, https://www.jacksonimmuno.com/ ) with BCIP/NBT (5-Bromo-4-chloro-3-indolylphosphate/Nitro-blue tetrazolium) Color Development Substrate (Bio-Rad, http://www.bio-rad.com/ ). A dilution series of the wild type sample was conducted to ensure that the evaluation of proteins was carried out within the limits of detection of the method and below the saturation levels of proteins.
10
Synthesis of Ni-NPs Using GFP-Tagged Proteins
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Nickel(ii ) chloride, CAS 7718-54-9, 98% (Sigma-Aldrich, USA), sodium borohydride, CAS 16940-66-2, 98% (Alfa Aesar, USA), polyvinylpyrrolidone (PVP), CAS 9003-39-8 (Sigma-Aldrich), green fluorescent protein (GFP-12xHis) with histamine tag (Huang Yuting), Streptococcus pneumoniae ATCC 49136 (Master Zhang Jiayu), Streptococcus mutans ATCC 25175 (Master Zhang Jiayu), Streptococcus pyogenes ATCC 12344 (Master Zhang Jiayu), Rabbit anti-SMU290 antibody (Zhang Jiayu), SureBlueTM TMBMicrowell peroxidase substrate (1-Component) (No. 52-00-00, KPL), Ni Sepharose 6 Fast Flow (No. 17-5318-01, GE Healthcare Life Sciences, USA), Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (No. 111-035-003, Jackson ImmunoResearch, USA), AffiniPure goat anti-rabbit IgG (H + L) (No. 111-005-003, Jackson ImmunoResearch), ChromPure mouse IgG, whole molecule (No. 015-000-003, Jackson ImmunoResearch), Streptococcus pneumoniae antibody: anti-SMU290 antibody from rabbit (Zhang Jiayu), pRSET_Gβ1 Escherichia coli (E. coli) BL21 (DE3) colony (Huang Yuting Xueyu), pRSET_SMU290 E. coli BL21 (DE3) colony (Zhang Jiayu), reagents for the culture solution (Zymeset, Sigma, Merck, Germany), and reagents for the buffer (Sigma, Lianhe Company, USA), FAS (Evonik, Germany) were used as precursors without any further purification. Argon was used as the carrier gas.
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