Whole cells and kidney glomeruli from five kidney tissues of 5 rats were mixed and extracted using the radio-immunoprecipitation assay buffer in the presence of protease inhibitors (Roche, Mannheim, Germany) at 4 ° C. Protein concentrations were measured using a Direct Detect Spectrometer (Merck, Darmstadt, Germany); 30 μg of proteins were electrophoresed by 15% SDS-PAGE as reducing conditions and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk in TBST for 1 h, the membranes were probed with the diluted primary rabbit antibodies against NLRP3 (ab214185, dilution 1: 500; Abcam, Shanghai, China), caspase-1 (ab1872, dilution 1: 1,000; Abcam), IL-1β (ab9722, dilution 1: 1,000; Abcam), and GAPDH (ab9485, dilution 1: 2,000; Abcam) overnight at 4 ° C. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibodies (ab97051, dilution 1: 5,000; Abcam) for 2 h at room temperature and visualized using horseradish peroxidase-conjugated secondary antibody with enhanced chemiluminescence substrate on a BioRad Chemidoc MP system (Bio-Rad). ImageJ software was used to quantify the immunoblots. The fold-changes of the target proteins were normalized to that of GAPDH. All Western blots were repeated 3 times and means of normalized densities were calculated.
Ab9722
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab9722 is a primary antibody that targets the protein RPS6. It is a rabbit polyclonal antibody produced using a synthetic peptide corresponding to a region within the N-terminal amino acids 1-50 of the human RPS6 protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab6671, by Abcam (34 mentions)
Ab214185, by Abcam (17 mentions)
Ab1793, by Abcam (12 mentions)
Ab179515, by Abcam (12 mentions)
Ab9324, by Abcam (11 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Ab1872, by Abcam (10 mentions)
Ab16502, by Abcam (9 mentions)
Ab4207, by Abcam (8 mentions)
Ab7737, by Abcam (8 mentions)
Bca protein assay, by Thermo Fisher Scientific (8 mentions)
Ab205718, by Abcam (7 mentions)
Ab137550, by Abcam (6 mentions)
Ab8227, by Abcam (6 mentions)
Ab6672, by Abcam (6 mentions)
Anti il 1β, by Abcam (5 mentions)
Ab39012, by Abcam (5 mentions)
Ab86299, by Abcam (5 mentions)
Ab263899, by Abcam (5 mentions)
Ab5076, by Abcam (5 mentions)
217 protocols using ab9722
1
Western Blot Analysis of NLRP3 Inflammasome
2
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue was homogenized, lysed and centrifuged at 2000 rpm for 20 mins. The supernatants were collected and protein concentrations were measured using BAC kits (Keygen Biotech, Nanjing, People’s Republic of China). Proteins were separated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (25 µg/lane), and membranes prepared by Western blot procedure were finally analyzed using the ECL system (Millipore, Bedford, USA). Primary antibodies were used as follows:
GAPDH (#8884, 1:10000, Cell Signaling Technology, Boston USA), Caspase-3 (#9662, 1: 1000, Cell Signaling Technology, Boston USA), Cleaved Caspase-3 (#9664, 1:1000, Cell Signaling Technology, Boston USA), Bax (#5023, 1:1000, Cell Signaling Technology, Boston USA), Bcl-2 (#15071, 1:1000, Cell Signaling Technology, Boston USA), HIF-1α (#36169, 1:1000, Cell Signaling Technology, Boston USA), VEGF (ab52917, 1:1000, Abcam, England), TNF alpha (ab1793, 1:1000, Abcam, England), IL-1 beta (ab9722, 1:2000, Abcam, England).
GAPDH (#8884, 1:10000, Cell Signaling Technology, Boston USA), Caspase-3 (#9662, 1: 1000, Cell Signaling Technology, Boston USA), Cleaved Caspase-3 (#9664, 1:1000, Cell Signaling Technology, Boston USA), Bax (#5023, 1:1000, Cell Signaling Technology, Boston USA), Bcl-2 (#15071, 1:1000, Cell Signaling Technology, Boston USA), HIF-1α (#36169, 1:1000, Cell Signaling Technology, Boston USA), VEGF (ab52917, 1:1000, Abcam, England), TNF alpha (ab1793, 1:1000, Abcam, England), IL-1 beta (ab9722, 1:2000, Abcam, England).
3
Proteolytic Cleavage of Inflammatory Mediators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human IL-1β, NLRP-3 or PYCARD (ASC) (280 ng, H00003553-P02, H00114548-P01 or H00029108-P01, Abnova) were incubated with recombinant active human MMP-7 (0.035U, #444270 Merck Millipore) in MMP reaction buffer (20 mM Tris, pH 7.6, 5 mM CaCl2, 0.1 M NaCl) at 37°C until stopped with 100 mM DDT. Fragments were detected by Western blot, using rabbit anti-IL-1 beta (1:2 000, ab9722, Abcam), rabbit anti-ASC (1:1 000, p9522-75, US Biological) and rabbit anti-NLRP3 (1:500, sc-66846, Santa Cruz).
4
Histological Analysis of Bladder Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were embedded in O.C.T. compound (VWR) and 5-μm-thick fresh cryosections on positively charged microscope slides (Superfrost/Plus; Thermo Scientific) were fixed with 4% paraformaldehyde or acetone-methanol (1:1 v/v). For H&E or immunohistochemistry, sections were blocked and permeabilized (0.2% Triton X-100, 5% goat normal serum (DAKO) or 1% BSA (Sigma), stained (anti-neutrophil antibody [NIMP-R14] (ab2557, Abcam), polyclonal E. coli antibody (1:100, NB200-579, Novus Biologicals), anti-IL-1 beta (1:50, ab9722, Abcam) or anti-MMP-7 (1:100, ab4044, Abcam), all rabbit antibodies). Alexa 488 anti-rat IgG or anti-rabbit IgG and Alexa 568 anti-rabbit IgG (A-21210, A-11001 and A-11011, Life Technologies) were secondary antibodies and nuclei were counterstained with DAPI (0.05 mM, Sigma-Aldrich). Imaging was by fluorescence microscopy (AX60, Olympus Optical). Richard-Allan Scientific Signature Series Hematoxylin 7211 and Eosin-Y 7111 (Thermo Scientific) were used to counterstain the tissue sections.
Histology was scored using H&E stained bladder sections. The score was based on neutrophil infiltration, tissue architecture and epithelial thickness on a scale of 0–10, where 0 is unchanged compared to uninfected controls and 10 the highest neutrophil infiltration, most destroyed tissue architecture and maximum epithelial thickness.
Histology was scored using H&E stained bladder sections. The score was based on neutrophil infiltration, tissue architecture and epithelial thickness on a scale of 0–10, where 0 is unchanged compared to uninfected controls and 10 the highest neutrophil infiltration, most destroyed tissue architecture and maximum epithelial thickness.
5
Immunofluorescent Analysis of Inflammasome Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were infected, fixed (3.7% formaldehyde, 10 min), permeabilized (0.25% Triton X-100, 5% FBS, 15 min), blocked (5% FBS, 1h at RT), incubated with primary antibodies in 5% FBS overnight at 4°C (anti-IL-1 beta, 1:100, ab9722; anti-MMP7, 1:25, ab4044, all Abcam; anti-ASC, 1:50, sc-22514-R, Santa Cruz; anti-NLRP3/Cryo-2, 1:100, AG-20B-0014-C100, Adipogen) and appropriate secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG, A-11034, or goat anti-mouse IgG, A-11001; Life Technologies), (1h at RT). After nuclear staining (DRAQ5, Abcam), slides were mounted (Fluoromount, Sigma-Aldrich), imaged by laser-scanning confocal microscopy (LSM510 META confocal microscope, Carl Zeiss) and quantified by ImageJ software 1.46r (NIH).
6
Protein Expression and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics) and fractionated using the NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific). Supernatants were filtered and concentrated by trichloroacetic acid precipitation, followed by aceton desiccation. Proteins were run on SDS-PAGE (4–12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes (GE Healthcare) blocked with 5% bovine serum albumin (BSA) or non-fat dry milk (NFDM), incubated with primary antibody: rabbit anti-IL-1 beta (1:2,500 in 5% NFDM, ab9722, Abcam), rabbit anti-ASC (1:200 in 5% BSA, sc-22514-R, Santa Cruz), mouse anti-NLRP3/NALP3 (1:1,000 in 5% milk, Cryo-2, Adipogen) or rabbit anti-MMP7 (1:200 in 5% BSA, ab4044, Abcam), washed with PBS tween 0.1% and incubated with secondary antibodies in 5% NFDM (goat anti rabbit-HRP or goat anti-mouse-HRP, Cell Signaling). Bands were imaged using ECL Plus detection reagent (GE Health Care) and quantified using ImageJ. GAPDH (1:1,000, sc-25778, Santa Cruz) was used as loading control.
7
Immunohistochemistry for Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed as previously described [36 (link)]. The paraffin sections were incubated with the following primary antibodies at 4 °C for 24 h: anti-Ki67 (1:400; Rabbit. no. ab15580; Abcam, Cambridge, UK), anti-TNF-α (1:100; Rabbit. no. ab9739; Abcam, UK), anti-IL-6 (1:500; rabbit. no. NB600-1131; Novus Biologicals, Littleton, CO, USA), anti-IL-1β (1:100; Rabbit. no. ab9722; Abcam, UK), and anti-MPO (1:100; Rabbit. no. ab208670; Abcam, Cambridge, UK). The sections were then incubated with the goat anti-rabbit IgG secondary antibodies (1:500; Rabbit. no. ab6112-HRP; Abcam, Cambridge, UK) at room temperature for 30 min. The sections were counterstained with hematoxylin and mounted on the slides. Each staining per specimen was repeated three times. All sections were observed using a digital microscope (DM2500; Leica, Wetzlar, Germany).
8
Western Blot Analysis of Penumbra
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For WB analysis, we used peri-infarct (penumbra) cortical regions. Using a brain matrix, the brains were rapidly dissected into 4.0 mm coronal sections (approximately 0.5 mm and −3.5 mm from bregma). Brain tissue was homogenized and processed for western blotting as previously described. Fifty-microgram of proteins were loaded in each lane and separated followed by transfer to nitrocellulose membranes. The membranes were blocked for non-specific binding and probed with primary antibodies against NLRP3, Caspase-1, ASC (1:1000; AG-20B-0014; AG-20B-0042; AG-25B-0006 Adipogen life sciences), cleaved IL-1β, Caspase-3, Phospho- NFκBp65, NFκBp65 (1:1000; CST-12242; 9664; 3033; 8242; Cell signaling technology), Cleaved PARP, Anti IL-18, IL-1β (1:1000; ab32064; ab71495; ab9722; Abcam, USA), phospho IκB (1:1000; Santa cruz biotechnology, SC8404) at 4 °C for overnight. Following TBS-T washes, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (1: 10,000; Sigma). The bands were then visualized by means of an enhanced chemiluminescent substrate system (Thermo fisher scientific). Protein levels were analyzed densitometrically, using Image J software and were normalized to loading controls, and expressed as fold change.
9
Hippocampal Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hippocampal tissue was weighted and washed with pre-cold PBS before lysis in RIPA buffer (no. P0013B, Beyotime, China) containing proteinase and phosphatase inhibitors (Sigma, USA), then homogenized using an ultrasonic crusher. The lysates were centrifuged at 12000rpm and 4 ℃ for 20 min. Bicinchoninic acid (BSA) method was performed to detect the protein concentration. The protein (30 ug) was separated by electrophoresis in 10% SDS-PAGE gels (KayGen Biotech, Co., Ltd), then transferred to polyvinylidene di uoride (PVDF; Bio-Rad Laboratories, USA) membranes. The PVDF membranes were incubated with 5% nonfat milk for 1 h for blocking nonspeci c binding reactions. These primary antibodies were used for incubating membranes overnight at 4°C: TREM2 (13483-1-AP, proteintech,1:1000), CD163(ab182422,abcam,1:1000),IL-1β(ab9722,abcam,1:1000),IL-6(ab229381,abcam,1:1000),actin (bs-0061R, Bioss, 1:20000). On the next day, the membranes were washed with TBST for three times and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Finally, the protein bands were detected using a chemiluminescence kit (ECL; Pierce, Illinois, USA) and analyzed via ImageJ.
10
ELISA Assays for Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ELISA kits utilized for evaluation of supernatant of homogenized NP tissues and cell supernatant included rat NLRP3 (15.63–1000 pg/mL, ab277086, Abcam), rat IL-1β (0.2–0.4 μg/mL, ab9722, Abcam), rat IL-18 (15.6–1000 pg/mL, ab312909, Abcam), rat gasdermin-D (0.156–10 ng/mL, #EKU09199, Biomatik, Delaware), rat BMP-7 (15.63–1000 pg/mL, NBP2-70002, Novus Biologicals).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!