RAW 264.7 macrophages were cultured in 24-well plates and treated with 30 µM of ZEA in the presence or absence of 700 nM 4-PBA for 24 h. Immunofluorescence staining of GRP78 and CHOP was performed. RAW 264.7 macrophages were first fixed in 4% paraformaldehyde for 20 min, and then permeabilized with 0.1% Triton X-100 in PBS for 20 min. They were subsequently blocked with 5% BSA in PBS for 1 h at room temperature. The membranes were then incubated with the chosen antibodies, including antibodies to CHOP and GRP78, at a dilution of 1:50 in TBST for 2 h at 37 °C. After washing followed by incubation with anti-rabbit secondary antibody (Invitrogen, A21206; 1:500 dilution) and anti-goat secondary antibody (Invitrogen, A21432; 1:500 dilution) for 1 h at 37 °C, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The fluorescent signals were examined under a Nikon epifluorescence microscope (Nikon Eclipse 80i, Nikon, Tokyo, Japan).
Anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The anti-rabbit secondary antibody is a laboratory reagent used in various immunoassay techniques, such as Western blotting and immunohistochemistry. It is designed to bind to primary antibodies raised against rabbit antigens, allowing for the detection and visualization of target proteins or molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Anti flag, by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Sirt3, by Cell Signaling Technology (2 mentions)
Hsp60, by BD (2 mentions)
Sirt3, by Merck Group (2 mentions)
Mtco1, by Abcam (2 mentions)
Foxo3a, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence ecl method, by GE Healthcare (2 mentions)
A21432, by Thermo Fisher Scientific (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
58 protocols using anti rabbit secondary antibody
1
Evaluating ER Stress Markers in Macrophages
2
Quantifying Liver Protein Expression
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Frozen liver sections were permeabilized using 0.3% Triton-X and incubated in antigen retrieval solution (Antigen retrieval citrate, Biogenex) at sub-boiling temperature for 10 min. Subsequently, sections were incubated with blocking buffer containing 5% normal donkey serum (Jackson Immuno Research) followed by incubation overnight at 4 °C with mouse anti-HNF4α monoclonal antibody (1:800, Cat# PP-H1415–00, R&D Systems), rabbit polyclonal anti-VDAC antibody (1:400, cat# PA1–954A, Invitrogen) and cleaved caspase3 antibody (1:500, cat#9664, Cell Signaling). Sections were washed and incubated for 1 h at room temperature with anti-mouse secondary antibody coupled with Alexa fluor 488 (1:400, Invitrogen) and anti-rabbit secondary antibody coupled with rhodamine red. Nuclei were visualized by counterstaining with DAPI (40,6-diamidino-2-phenylindole, Sigma Aldrich). Slides were mounted using fluorescence mounting medium and images were obtained at ×40 magnification using an Olympus IX71 fluorescence microscope. Fluorescence intensity of HNF4α-stained nuclei and VDAC stained mitochondria was calculated using MetaMorph TL software (version 7.6.5.0, Olympus). Fold change was calculated by normalizing to values from mice fed NC.
3
Immunohistochemical Analysis of Autophagy Markers
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The expression of Beclin-1, LC3, and 4E-BP1 in primary tumors was detected through immunohistochemical staining (IHC) and then compared with the expression in the normal tissue samples. The formalin-fixed paraffin-embedded tissue was cut into 4-μm thick sections, dewaxed, rehydrated, and blocked with hydrogen peroxide. After incubation with primary antibodies against 4E-BP1 (1:200), Beclin-1 (1:100), and LC3 (1:400) (all from Cell Signaling Technology, United States) diluted in 1% PBS, the sections were incubated with an anti-rabbit secondary antibody (Invitrogen, United States) for 30 min. After an additional two washes in PBS, the sections were incubated with diaminobenzidine (DAB, Invitrogen, United States) for 10 min to detect signals. Finally, the sections were counterstained with hematoxylin (Invitrogen, United States). Negative controls were obtained by substituting nonimmune rabbit serum for the primary antibodies.
4
Western Blot Analysis of Heat Shock Proteins
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Proteins were transferred to PVDF membrane, blocked in blocking buffer (5% w/v skim milk powder in PBS-T) for 1 h at room temperature and then incubated with primary antibody in blocking buffer. The following antibodies were incubated overnight at 4 °C: anti-HSPA1A (Origene, cat #TA500772, 1:10,000), anti-HSF1 (Abcam, cat #ab52757, 1:40,000), anti-BAG1 (Abcam, cat #ab32109, 1:750), and anti-myc (Thermofisher, cat #13–2500, 1:1,000). The blots were washed in PBS-T and then incubated with either anti-rabbit secondary antibody (Invitrogen, cat #65-6120, 1:20,000) or anti-mouse secondary antibody (Invitrogen, cat #31430, 1:20,000) in PBS-T for 1 h at room temperature. Proteins were detected by an enhanced chemiluminescence kit (Clarity, BioRad). The uncropped blots are shown in Supplementary Fig. 7 .
5
Immunofluorescence Analysis of CD3+ T Cells
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Tumour tissues were dissected, embedded in tissue freezing medium, and frozen sectioned using freezing microtome (Lecia CM1900, Germany). 10 μm sections fixed in acetone were rinsed with PBS, and were blocked with 2% BSA followed by incubation with rabbit-anti-CD3 (Abcam, 1:100 diluted) at 4 °C overnight. Then sections were incubated with anti-rabbit secondary antibody (Invitrogen, 1:200 diluted) for 1 h at 37 °C. Nuclei were stained with 1 μg/ml DAPI (Sigma). The images were captured using a confocal fluorescence microscope (Olympus) and were analyzed by using FV10-ASW software.
6
Transthyretin Detection in Seeding Reactions
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The presence of transthyretin in the elutions from extractions and the incorporation of MTTR into the seeding reaction was confirmed by dot-blot analysis.
The insoluble fraction of the seeding reaction was obtained by spinning the samples for 1 hour at 13000xg.The pellet was resuspended in 6M of Guanidine HCl.
5 μL of the final extraction elutions or the resuspended pellet were blotted onto a nitrocellulose membrane (0.2 μm; Bio-Rad). The transthyretin from the extractions was observed by using anti-transthyretin antibody (1:1000, Genscript) and anti-rabbit secondary antibody (1:1,000; Invitrogen 31460). The presence of MTTR in the pellet was visualized by using HisProbe (Thermo Scientific Fisher) and HRP antibody (1:5000).
The insoluble fraction of the seeding reaction was obtained by spinning the samples for 1 hour at 13000xg.The pellet was resuspended in 6M of Guanidine HCl.
5 μL of the final extraction elutions or the resuspended pellet were blotted onto a nitrocellulose membrane (0.2 μm; Bio-Rad). The transthyretin from the extractions was observed by using anti-transthyretin antibody (1:1000, Genscript) and anti-rabbit secondary antibody (1:1,000; Invitrogen 31460). The presence of MTTR in the pellet was visualized by using HisProbe (Thermo Scientific Fisher) and HRP antibody (1:5000).
7
Protein Extraction and Analysis from Plant Cells
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Cells were pelleted by centrifugation at 3500 × g for 5 min at 4°C, washed with PBS and resuspended in 50 mM Tris pH 8, SDS 9%, PID, PMSF 0.1 M and NaCl 150 mM. Total protein extracts were obtained by freeze/thaw cycles in liquid nitrogen followed by another mechanical disruption procedure as described above. Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Comassie blue, and transferred onto a PVDF membrane (Immobilon ®-P, pore size 0.45 μm) using a power blotting station (Invitrogen™, Thermo Fischer). Immunoblotting analysis was performed with an antibody against photosystem II subunit S (PsbS) at 1:1000 (Agrisera). Anti-rabbit secondary antibody was used at 1:10,000 (Invitrogen). Immunoblots were visualized using IQ800 Control software (ImageQuant 800, Amersham).
8
Neuroanatomy Analysis of Mouse Brain
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The mice were anesthetized with 20% urethane (10 ml/kg, i.p.). The brains were fixed in 4% paraformaldehyde overnight and then dehydrated in 20 and 30% sucrose until they sunk. The tissues were cut into 15-μm-thick coronal sections. The sections were stained with neuronal nuclear protein (NeuN) antibody (1:400, CST, United States) overnight at 4 °C and incubated with an anti-rabbit secondary antibody (1:500, Invitrogen, United States) for 2 h at room temperature. Images were captured by using the Nikon microscope (Eclipse Ci-E, JP).
9
Immunohistochemistry of Cardiac Tissue
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For immunohistochemistry, hearts were snap frozen in liquid nitrogen, prior to embedding in OCT. 6–10 μm sections were cut using a cryostat (Leica), air dried and fixed in 4% PFA, (paraformaldehyde) in PBS or in acetone, at -20°C for 15 min, followed by washing in 0.1% PBS-Triton X-100 [14 (link)]. Blocking was achieved by incubation with 5% BSA-C (Aurion) in 0.1% PBS-Triton X-100, for at least 30 min at RT. Immunolabelling with primary antibodies (anti-collagen VI 1 in 100 (600-401-108-05, Rockland); anti-vinculin 1 in 200 (V4505, Sigma) was performed in 0.1% PBS-Triton X-100, 1% BSA-C, overnight in a humidified box at 4°C. Sections were washed 3 times in PBS, incubated for 60 min at RT in a dark box, with an anti-rabbit secondary antibody (FITC Invitrogen, 1:1000 in PBS), washed 3 times in PBS and counterstained with phalloidin (Sigma) and DAPI or draq5 (Invitrogen). Sections were mounted in Vectashield mounting medium (Vector Laboratories). Sections were examined using the Leica TCS SP4 laser scanning confocal microscope and analysed with Leica Application Suite (LAS) v5 (Leica Microsystems, Heidelberg, Germany).
10
dTAG-47 Chromatin Immunoprecipitation
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