Immunohistochemistry (IHC) staining was performed according to a previous method [13 (link), 20 ]. Serial paraffin sections were subjected to antigen retrieval, incubation in antigen retrieval solution for 20 min, inactivation with endogenous peroxidase (3% H2O2), and blocked in goat serum for 1 h. Sections were then incubated with primary antibodies against CD3 (SC-20047, Santa Cruz Biotechnology Inc., Dallas, TX, USA), F4/80 (SC-377009, Santa Cruz Biotechnology Inc.), IL-6 (GeneTex, Santa Cruz Biotechnology Inc.), IL-1β (ab9722, Abcam), TNF-α (ab183218, Abcam), p-p65 (Ser536) (#3033, Cell Signaling Technology), p16 (ab211542, Abcam), NLRC4 (ab201792, Abcam), ITGAM (ab133357, Abcam), p19 (ab80, Abcam), Collagen I (14695-1-AP, Proteintech), Collagen III (22734-1-AP, Proteintech), α-SMA (14395-1-AP, Proteintech), and β-galactosidase (15518-1-AP, Proteintech). After washing, sections were incubated with a secondary antibody for 1 h, and processed using the SABC-POD kit (SA2001, Boster, China). Then, sections were counterstained with Hematoxylin and fixed with biomount medium. Hematoxylin and Shandon Instant Eosin (Solarbio Co., Ltd.) were used to determine cell infiltration. Masson's trichrome staining (Sigma-Aldrich®) was used to assess collagen deposition.
Ab183218
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab183218 is a laboratory product offered by Abcam. It is a general-purpose lab equipment designed for researchers and scientists. No further details about the core function or intended use of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab254360, by Abcam (11 mentions)
Ab259341, by Abcam (9 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Ab8245, by Abcam (7 mentions)
Ab16502, by Abcam (6 mentions)
Ab233706, by Abcam (6 mentions)
Ab9722, by Abcam (5 mentions)
Ab32503, by Abcam (5 mentions)
Ab205718, by Abcam (4 mentions)
Ab32124, by Abcam (4 mentions)
Ab179800, by Abcam (3 mentions)
Ab52894, by Abcam (3 mentions)
Ab216995, by Abcam (3 mentions)
Ab9485, by Abcam (3 mentions)
Ab263899, by Abcam (3 mentions)
Ab7817, by Abcam (3 mentions)
Ab178945, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ab184787, by Abcam (3 mentions)
Ab32536, by Abcam (3 mentions)
57 protocols using ab183218
1
Comprehensive Immunohistochemical Profiling
2
Western Blot Analysis of Inflammatory Markers
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Proteins extracted from BEAS-2B cells and HUVECs were measured using a bicinchoninic acid kit (Beyotime Biotechnology, China). Then, the proteins were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The PVDF membranes were incubated using 5% skimmed milk, and then with primary antibodies at 4 °C overnight. Blots were probed using the following antibodies: anti-IL-1β (1: 1, 000, ab234437; Abcam, Cambridge, UK), anti-TNF-α (1: 1, 000, ab183218; Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1: 2, 000, bs0755R; Bioss, China), with GAPDH being the endogenous control. Then, membranes were further incubated for 1 h using a secondary antibody (1:2, 000, b-0311P-HRP; Bioss).
3
Immunohistochemical Analysis of Corneal Structure
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The whole eyeballs were immersed in 4% paraformaldehyde for 24 h at room temperature. Subsequently, the eyeballs were treated with gradient alcohol and xylene and embedded in paraffin for sectioning at a thickness of 4 μm. Tissue sections stained with hematoxylin/eosin were used to observe the corneal structure. Immunohistochemical staining was performed according to our previously published protocol52 (link). Briefly, antigen retrieval was performed with sodium citrate repair solution by microwave heating, non-specific antigens were eliminated by 3% H2O2, and the sections were blocked with goat serum for 1 h and incubated with primary antibodies against IL-1β (1:100, ab254360, Abcam), TNF-α (1:1000, ab183218, Abcam), TRAF6 (1:300, ab33915, Abcam), IRAK1 (1:1000, ab238, Abcam), and COX2 (1:400, ab179800, Abcam) overnight at 4 °C. Next day, the sections were incubated with the Envision horseradish peroxidase system (Gene Tech, Shanghai, China) for 2 h at room temperature. The sections were finally incubated with 3,3′-diaminobenzidine (DAB) (Gene Tech, Shanghai, China) for 5 min. The staining was observed and captured with an Eclipse Ni microscope (Nikon, Tokyo, Japan).
4
Protein Expression Analysis of EGFR, p-EGFR, and TNF-α
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The cornea and conjunctiva were lysed with cold radioimmunoprecipitation buffer (1% Triton X-100, 1% sodium deoxycholate. 0.1% sodium dodecyl sulfate, 0.15 M NaCl, 0.05 M Tris-HCl). Protein concentrations were measured using a BCA kit (MicroBCA; Pierce; Thermo Fisher Scientific, Inc.). Equal amounts (20 μl) of proteins were subjected to electrophoresis on 8% SDS-PAGE and then transferred into polyvinylidene difluoride membranes. The membranes were blocked with 2% BSA at room temperature for 1 h and then incubated with primary antibodies directed against EGFR (1:1,000; ab52894), p-EGFR (1:1,000; ab40815) or TNF-α (1:400; ab183218) (all from Abcam, Cambridge, MA, USA) and β-actin (1:10,000; A5441; Sigma-Aldrich; Merck KGaA) as a loading control at 4°C overnight, as previously described (15 (link)). The membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; 1706515; Bio-Rad Laboratories, Inc., Hercules, CA, USA) secondary antibodies at room temperature for 2 h. Signals were developed using enhanced chemiluminescence reagents (Xiamen Lulong Biotech Co., Ltd., Xiamen, China) and captured on film.
5
Immunoblotting Analysis of Inflammatory Markers
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Primary antibodies against MARCO (ab239369), TRIF (ab13810), NF‐κB (ab16502), TNF‐α (ab183218), Lamin B (ab133741) and GAPDH (ab181602) as well as the secondary antibody of goat anti‐rabbit were purchased from Abcam. Primary antibody against IκB‐α (4814) was purchased from Cell Signalling Technology. Primary antibody against Toll‐like receptor 4 (TLR4) (AF7017) was purchased from Affinity. PolyG (P4404) and LPS (L2880) was purchased from Sigma.
6
Quantifying Inflammatory Biomarkers Using ELISA
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ELISA kits were used to detect the expression levels of proinflammatory factors, including Il-6 (ab275971, Abcam), Il-8 (ab7747, Abcam), Tnf-α (ab183218, Abcam), Mcp-1 (ab9858, Abcam), Vcam-1 (ab223591, Abcam), and Icam-1 (ab174445, Abcam). The required plates were removed from the aluminum foil bag after equilibrating at room temperature for 20 min. Standards with different concentrations were added to the standard wells, and the standard curve was prepared by routine operation. Fifty microliters of the sample and horseradish peroxidase (HRP)-labeled detection antibodies were added to the sample well. The plates were sealed and shaken on a shaker for 3 h. After the routine washing operation, 100 mL of substrate solution was added to each test well and incubated at room temperature for 15 min. The stop reaction solution (100 μL) was added to stop the reaction. The OD value of each well was detected at 450 nm using a microplate reader.
7
Molecular and Cellular Mechanistic Study of Inflammation
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All the reagents were analytical grade and purchased from commercial providers. Lipopolysaccharide (LPS), 5-carboxyfluorescein (FAM) and PCR kit were purchased from Sigma Co., United States. Human chondrocyte CHON-001 and HUVEC (Human umbilical vein endothelial cells) were purchased from ATCC. TRIzol reagent was from Invitrogen. Using a reverse transcription kit, reverse transcription was carried out (TaKaRa Company, Dalian, China). Beyotime Biotechnology Co., was the source of the cell-counting Kit-8 (CCK-8). Luciferase activity was measured with a Luciferase detection kit (Shanghai Yisheng Biotechnology Co., LTD) according to the manufacturer’s protocol. The following primary antibodies were used: GAPDH (mouse mAb, Abcam #ab8245), IL-1 (rabbit mAb, Abcam #abab9722), IL-6 (mouse mAb, Abcam #ab9324), MCP-1 (rabbit mAb, Abcam #ab214819), TNF (rabbit mAb, Abcam #ab183218). Cell lysate aliquots were separated in 10% SDS-PAGE (Beyotime, Code NO. P0012A). Rabbit secondary antibody, mouse secondary antibody, and Lipofectamine® 3000 were purchased from Thermo Fisher Scientific. Deionized water (Millipore Milli-Q Complete) with a resistivity of 18.2 MΩ was used in all experiments. All the solutions were prepared with DEPC-treated water.
8
Protein Expression Analysis in Mouse Corneal Tissues
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Mouse corneal tissues or stromal cells were lysed using radioimmunoprecipitation lysis buffer to extract the proteins, and the total proteins were separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoresis, proteins were transferred to polyvinylidene fluoride membranes through Tris-glycine-SDS buffer and blocked. The main antibodies included Recombinant Anti-METTL3 (ab195352; Abcam), Anti-beta Actin (ab8227; Abcam), Akt Antibody (9272; Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (4060; Cell Signaling Technology), Recombinant Anti-PI 3 Kinase p85 alpha (ab191606; Abcam), Anti-PI 3 Kinase p85 alpha (phospho Y607) (ab182651; Abcam), Recombinant Anti-TNF alpha (ab183218; Abcam), Anti-IL-1 beta (ab9722; Abcam), and Anti-IL-6 (23431-1-AP; Proteintech). The membranes were incubated with the primary antibodies overnight. The primary antibody was recovered, and the membranes were rinsed three times with Tris-buffered saline with 0.1% Tween 20 and then incubated for 2 hours with a specific secondary antibody. Finally, the proteins were analyzed with enhanced chemiluminescence detection reagents for quantification (WBKLS0100; MilliporeSigma, Burlington, MA, USA).
9
Western Blotting Analysis of Inflammatory Markers
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Western blotting was performed using antibodies against calpain-2 (Cat. 2,539, 1:1,000; Cell Signaling Technology), IL-6 (ab233706,1:1,000; Abcam, Cambridge, United Kingdom), TNF-α (ab183218,1:1,000; Abcam, Cambridge, United Kingdom), IL-1β (ab216995,1:1,000; Abcam, Cambridge, United Kingdom), p-STAT3 (Tyr705) (Cat. 9,145, 1:1,000; Cell Signaling Technology), STAT3 (Cat. 5,345, 1:1,000; Cell Signaling Technology), and GAPDH (Cat. 2,118, 1:5,000; Cell Signaling Technology). Next, the proteins were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualised using a ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, United States). The relative quantity of proteins is presented as the ratio of target proteins to GAPDH.
10
Western Blot Analysis of Protein Expression
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Radio immunoprecipitation assay (RIPA) buffer containing the PMSF was used to dissolve brain tissues. After centrifugation, the supernatant was used for protein concentration detection. The same amount of protein was applied for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% skimmed milk (3 h). The membrane was washed with PBST, and incubated with related primary antibodies (1:800) overnight at 4°C. After washing with PBS twice, the membrane was incubated with second antibody (1:2000) for 3 h. Chemiluminescence with Thermo ECL Substrate (Bio‐Rad) was used to incubate the membrane, and ImageJ software was used to analyze the band gray. The antibodies used in this study were listed as follows: anti‐Bax antibody (ab32503; Abcam), anti‐nuclear factor erythroid 2‐related factor 2 (Nrf2) antibody (ab62352; Abcam), anti‐B‐Cell CLL/Lymphoma 2 (Bcl‐2) antibody (ab32124; Abcam), antitumor necrosis factor (TNF)‐α antibody (ab183218; Abcam), anti‐NLRP3 antibody (ab263899; Abcam), anti‐IL‐1β antibody (ab254360; Abcam), anti‐Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (ab9485; Abcam), and goat anti‐Rabbit IgG (ab205718; Abcam).
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