Cerebellum tissues were homogenized by sonication in 0.2 M perchloric acid (100 μM EDTA•2Na) and centrifuged at 20.000×g for 15 min. Supernatants were kept frozen until analysis. Tissue glutamate concentrations were measured by enzyme-linked immunoassay (ELISA). Ninety-six well plates were coated with cerebellum homogenates (10 μg/well) at 4°C overnight. After washing with PBS (phosphate buffered saline, 0.05% Tween 20), in each well, anti-glutamate antibody was added (rabbit, 1:1,000, ab37070, Abcam) and incubated for 2 hours at room temperature. Thereafter, the samples were treated with horseradish peroxidase-conjugated anti-rabbit antibody (1:1,000, Sigma-Aldrich) for 2 hours at room temperature and with substrate solution (R&D systems, Minneapolis, MN, USA) for 20 min. The optical density was measured at 450 nm with a micro-plate reader (SpectraMAX M5, molecular device, Sunnyvale, CA, USA) after stopping peroxidase response with a stop solution (R&D systems).
Horseradish peroxidase conjugated anti rabbit antibody
Manufactured by Merck Group
Sourced in United States, Belgium
Horseradish peroxidase-conjugated anti-rabbit antibody is a reagent used in immunoassays and other laboratory techniques. It consists of an anti-rabbit antibody chemically linked to the enzyme horseradish peroxidase. This conjugate is used to detect and quantify the presence of rabbit-derived proteins or antigens in various samples.
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Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Substrate solution, by R&D Systems (1 mentions)
Stop solution, by R&D Systems (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Enhanced chemiluminescence reaction, by Cytiva (1 mentions)
Anti il 1r1, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (1 mentions)
Recombinant rat il 1β, by R&D Systems (1 mentions)
H 153, by Santa Cruz Biotechnology (1 mentions)
Anti annexin 5 antibody, by Abcam (1 mentions)
Il 1ra, by R&D Systems (1 mentions)
Ab4753, by Abcam (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
96 well microtiter plate, by Greiner (1 mentions)
Nupage 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Imagequant las 500 camera, by GE Healthcare (1 mentions)
20 protocols using horseradish peroxidase conjugated anti rabbit antibody
1
Cerebellum Glutamate Quantification by ELISA
2
Quantifying Pneumococcal GAPDH in Cell Wall
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The amount of pneumococcal GAPDH in the cell wall compartment of the R6 wild-type strain grown in CY or in CY supplemented with 1% Cho and of the lytA mutant in CY was analyzed by cell fractionation. One-tenth of a 100-ml culture in late exponential growth phase (OD600nm 0.6) was centrifuged (15 min at 3,000 g), and the pellet was resuspended in 1 ml of PBS containing 100 μg/ml lysozyme and 50 U/ml mutanolysin and incubated for 2 h at 37°C. The lysate samples were submitted to SDS-PAGE and stained by Coomassie blue. The total amount of proteins in each sample was quantified using the ImageJ software in order to correct, if necessary, equivalent loads of samples. The remaining 90 ml were centrifuged (15 min, 3,000 g), the pellet was resuspended in 9 ml of PBS containing 100 μg/ml lysozyme, 50 U/ml mutanolysin, 30% sucrose and incubated for 2 h at 37°C. This lysate was centrifuged and the supernatant containing the cell wall was collected. The cell wall fractions were separated by SDS-PAGE, transferred on a nitrocellulose membrane and analyzed by Western blot using rabbit anti-pneumococcal GAPDH antibody (1:5,000 dilution), horseradish peroxidase-conjugated anti-rabbit antibody (1:5,000 dilution, Sigma Aldrich) and ECL (Pierce) as detection reagent. The intensity of the spots was quantified using the ImageJ software.
3
Lipid Raft Protein Immunoblotting
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Membrane lipid raft and non raft proteins (40 mg) were fractionated on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% dried skim milk or BSA, filters were then incubated overnight with anti-IL-1R1 (1/100 Santa Cruz Biotechnology). After three washings, membranes were incubated with a horseradish peroxidase–conjugated anti-rabbit antibody (diluted 1∶3000; Sigma-Aldrich). Immunoreactivity was detected using an enhanced chemiluminescence reaction (Amersham Biosciences, Little Chalfont, U.K) and analyzed with the chemiluminescence imager (Chemismart 5000, Fisher Bioblock, France).
4
Caspase-1 Detection in Cell Lysates
5
Rat Insulinoma Cell Immunofluorescence and Apoptosis
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INS-1 rat insulinoma β cells used in the study have been obtained by CB Wollheim [18] (link). For immunofluorescence studies, we used rabbit anti-cytokines antibodies against IL-1 β (H-153), anti-cytokine receptor antibodies against IL-1R1 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit (Vector Laboratories, Burlingame, CA) was used as secondary antibody. Ganglioside M1 (GM1) detection has been performed using Alexa 488 Fluor conjugated-Cholera toxin B subunit (Invitrogen). Western blot experiments were performed with rabbit anti-IL1-R1 (H-150) (Santa Cruz Biotechnology) and horseradish peroxidase–conjugated anti-rabbit antibody (Sigma-Aldrich) was used to detect the signal. We used peroxidase-conjugated cholera toxin B subunit (Invitrogen) for dot-blot experiments. For apoptosis detection, rabbit polyclonal anti-annexin V antibody (Abcam, Cambridge, UK) was used at 5 mg/ml. Recombinant rat IL-1β and IL-1Ra were purchased from R&D Systems (Minneapolis, MN).
6
Extraction and Detection of Atg8 Proteins
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Whole-cell proteins were extracted by sonicating a pellet of 250 ml of cells resuspended in RIPA buffer (25 mM Tris pH = 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) and centrifuging (500 g for 5 min at 4°C) to remove the cell debris. Virion proteins were extracted by adding an equal volume of RIPA buffer to concentrated virions and boiling the sample for 10 min. Proteins were separated on a 6 M urea sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes. Anti-Atg8 (raised against the yeast Atg8, Abcam, ab4753) and the secondary horseradish peroxidase-conjugated antirabbit antibody (Sigma-Aldrich) were diluted 1:4000 and 1:10 000, respectively, in Tris-buffered saline containing 0.1% Tween 20 and 5% milk powder. The ECL-Prime western blotting detection reagent (GE Healthcare) was used for detection. Note that the antibody cannot distinguish between the E. huxleyi Atg8a and Atg8b protein sequences.
7
Quantitative Immunoblotting of Hepatic Proteins
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Proteins from total hepatic total cell membranes were separated by SDS-PAGE and transferred to nitrocellulose membranes (BioRad Laboratories, Hercules, CA, USA) [29 (link)]. Immunoblotting was performed using a polyclonal rabbit anti–SR-BI antibody (1:3000 dilution) donated by Dr. Karen Kozarsky (SmithKline Beecham Pharmaceuticals, King of Prussia, PA, USA) [32 (link)] and an anti-LDLR antibody (1:2000 dilution) donated by Dr. Joachim Herz (University of Texas Southwestern Medical Center, Dallas, TX, USA). Polyclonal rabbit anti-ε-COP (1:5000 dilution) donated by Dr. Monty Krieger (MIT, Cambridge, MA, USA) [33 (link)] was used as a loading control. Membranes were then incubated with secondary horseradish peroxidase-conjugated anti-rabbit antibody (1:5000, Sigma-Aldrich, St Louis, MO, USA), and finally, protein/antibody complexes were detected by enhanced chemiluminescence and quantified by densitometry using Image J (National Institutes of Health, Bethesda, MD, USA).
8
Measuring C1q Binding to CR1 Variants
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96-well microtiter plates (Greiner Bio-One) were coated with 3.4 pmol of each CR1 variant in PBS, and incubated overnight at 4°C. Saturation was then performed by adding 250 µl of PBS containing 1% BSA and 0.05% tween 20 per well for 90 min at room temperature. Four washes were performed using 200 µl of PBS, 0.05% tween 20 (PBS-T). C1q (10 µg/ml in PBS-T) was added and incubated at room temperature for 90 min. After four washes, bound C1q was detected by successive incubations with an anti-C1q rabbit polyclonal antibody (1:1,000 dilution) and a horseradish peroxidase-conjugated anti-rabbit antibody (Sigma) (1:20,000 dilution). After extensive washes with PBS-T, 100 µl of tetramethylbenzidine solution (Tebu-Bio) was added to each well. The reaction was stopped by adding 100 µl of 1 M H2SO4 and the optical densiy at 450 nm of each well was measured using an ELISA plate reader (FLUOstar Optima; BMG Labtech).
9
Quantification of Pneumococcal GAPDH Exposure
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The amount of surface-exposed pneumococcal GAPDH was analyzed by an alkaline elution strategy as described previously [16 (link)]. Briefly, a 50-ml culture in late-exponential growth phase (OD600nm 0.6) was harvested by centrifugation (15 min at 3,000 g). The cells were resuspended in 100 mM carbonate (pH 10) buffer and incubated for 30 min at 37°C. After centrifugation (15 min at 11,000 g), the pellet (cytoplasmic extract) and the alkaline supernatant (surface-associated fraction) were collected. Proteins were separated by SDS-PAGE, transferred on a nitrocellulose membrane and analyzed by Western blot using rabbit anti-pneumococcal GAPDH serum (1:5,000 dilution), horseradish peroxidase-conjugated anti-rabbit antibody (1:5,000 dilution, Sigma Aldrich) and ECL (Pierce) as detection reagent. The intensity of the spots was quantified using the ImageJ software. The amount of GAPDH in each sample was determined related to the GAPDH released by the wild-type strain. Pneumococcal FtsZ was detected using polyclonal rabbit serum.
10
Immunoblotting for CnoX Protein
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