Soft palate tissues were harvested from OSA and control groups, then fixed with paraformaldehyde and embedded in paraffin. For histology, paraffin sections (4 μm thick) were stained with hematoxylin and eosin. For immunohistochemistry, paraffin sections were de‐paraffinized, rehydrated and antigen retrieved with incubation in 0.01 m citrate buffer (pH 6.0) at 121 °C per 100 kpa for 2 min. Slides were incubated with primary antibodies including anti‐CD68 (dilution 1 : 200; ab125212; Abcam, Cambridge, UK), anti‐HMGB1 (dilution 1 : 200; ab18256; Abcam), anti‐TLR4 (dilution 1 : 200; GB11519; Servicebio, Woburn, MA, USA) and anti‐CD31 (dilution 1 : 200; ab28364; Abcam) at 4 °C overnight. Then secondary antibody conjugated with fluorescent dye were incubated at 37 °C for 30 min. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich, St Louis, MO, USA). Images were acquired with a 50i Nikon fluorescence microscope (Nikon, Tokyo, Japan) and processed with photoshop cs 4 software (Adobe Sytems, San Jose, CA, USA).
Gb11519
Manufactured by Wuhan Servicebio Technology
Sourced in United States
GB11519 is a piece of laboratory equipment. It is used for analytical purposes in scientific research and testing. The core function of this product is to perform specific analysis and measurement tasks within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab125212, by Abcam (2 mentions)
Ab18256, by Abcam (2 mentions)
Ab28364, by Abcam (1 mentions)
50i fluorescence microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab233706, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Total protein extraction kit, by Merck Group (1 mentions)
Nlrp3, by Abways (1 mentions)
Cy5651, by Abways (1 mentions)
Bs 0465r, by Bioss Antibodies (1 mentions)
G1211, by Wuhan Servicebio Technology (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Wuhan Servicebio Technology (1 mentions)
Abs118047, by Absin (1 mentions)
T0004, by Affinity Biologicals (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Ba0362, by Boster Bio (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Gb11131, by Wuhan Servicebio Technology (1 mentions)
10 protocols using gb11519
1
Histological and Immunohistochemical Analysis of Soft Palate Tissues in OSA
2
Protein Expression Analysis in Soft Palate
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Total proteins from soft palate tissues were extracted with a Total Protein Extraction Kit (Merck Millipore, Burlington, MA, USA) in accordance with the manufacturer's instructions . Proteins were loaded onto 10% SDS/PAGE and then transferred to poly(vinylidene difluoride) membranes (Merck Millipore). The transferred membranes were blocked in 5% BSA for 1 h at room temperature. Membranes were incubated at 4 °C overnight with primary antibodies against HMGB1 (dilution 1 : 2000; ab18256; Abcam), CD68 (dilution 1 : 1000; ab125212; Abcam), CD31, TLR4 (dilution 1 : 1000; GB11519; Servicebio), p‐NF‐κB p65 (dilution 1 : 2000; 8214S; Cell Signaling Technology, Danvers, MA, USA), VEGF (dilution 1 : 2000; 500661S; Cell Signaling Technology), MMP9 (dilution 1 : 2000; 13667S; Cell Signaling Technology), IL‐6 (dilution 1 : 2000; ab233706; Abcam) and GAPDH (dilution 1 : 10 000; 5174S; Cell Signaling Technology). Appropriate horseradish peroxidase‐conjugated secondary antibodies were incubated at 37 °C for 30 min. Finally, protein bands were detected with a chemiluminescent substrate.
3
Quantifying TLR4 and NLRP3 Expression
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4
Immunohistochemical Analysis of TLR4, MyD88, and NF-κB p65
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Immunohistochemical analysis was performed using anti-TLR4 (1:1000, GB11519, Servicebio), anti-MyD88 (1:200, GB11269, Servicebio), and anti-NF-κB p65 (1:400, bs-0465R, Bioss) antibodies. Fixed tissue gradient alcohol dehydration, paraffin embedding, and slicing to a thickness of 4 μm were performed. After deparaffinization and rehydration, the tissue sections were antigen-repaired with citric acid antigen-repair buffer (pH 6.0) and heated for the specified time. After natural cooling, the slides were placed in PBS (pH 7.4) and washed by shaking on a decolorization shaker three times for 5 min each. After treatment with 3% H2O2 and incubation at room temperature in the dark for 25 min, the slides were placed in PBS (pH 7.4) and washed by shaking on a decolorization shaker 3 times for 5 min each. Subsequently, 3% BSA was added to the circle to cover the tissue evenly, and the tissues were sealed for 30 min at room temperature. After gently removing the sealing solution, the sections were placed flat in a wet box with primary antibodies and incubated overnight at 4°C, followed by incubation with secondary antibodies for 30 min. The sections were then stained with diaminobenzidine (DAB) (G1211, Servicebio) and hematoxylin and mounted with neutral balsam. Brownish-yellow granules indicated positive signals. The slides were scanned and evaluated using Image-Pro Plus software.
5
Western Blot Analysis of Macrophage Proteins
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Total protein was extracted from BMDMs in radioimmunoprecipitation lysis buffer with a complete protease-inhibitor cocktail (Servicebio). Protein concentrations were detected using a BCA protein assay kit (Beyotime Biotechnology, Beijing, China). Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals). The membrane strips were then stained by incubating with an appropriate horseradish peroxidase-conjugated secondary antibody for 2 h at 25°C and visualized by enhanced chemiluminescence (Millipore, Billerica, MA, USA). ImageJ software (National Institutes of Health) was used to quantify the relative densities of proteins, which were normalized against GAPDH. All experiments were performed in triplicate.
6
Immunohistochemical Analysis of LC3, TLR4, and MMP3
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Immunohistochemical investigations were carried out using indirect method of peroxidase with a primary antibody specific for LC3 (anti-LC3, GB11124, Servicebio, Wuhan servicebio technology CO., LTD), TLR4 (anti-TLR4, GB11519, Servicebio), and matrix metallopeptidase 3 (MMP3, anti-MMP3, GB11131, Servicebio). The protocols of immunohistochemiscal investigation were the same as our previous study (Yu et al., 2023 (link)).
7
LPS-induced NF-κB signaling pathway
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LPS was obtained from Solarbio (055: B5, Beijing, China). The antibodies against MyD88 (23230-1-AP), NF-κB p65 (10745-1-AP), and phospho-NF-κB p65 were obtained from Proteintech (Hubei, China). The antibodies against TRL4 (GB11519) and β-actin were gained from Servicebio (Hubei, China). All secondary antibodies used for western blot were purchased from ImmunoWay (Plano, TX). Cy3-conjugated goat anti-rabbit IgG (H + L) used in immunofluorescence experiments was obtained from Servicebio (Hubei, China).
8
Immunohistochemical Profiling of Inflammation Markers
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The sections were microwave antigen‐retrieved in citrate solution. For immunohistochemistry (IHC), commercial IHC kits (KIT‐9707; Maixin) were used according to the manufacturer's specifications. Endogenous peroxidase activity and nonspecific binding were blocked. Then the sections were incubated with primary antibodies against HMGB1 (1:300; EPR3507; Abcam), RAGE (1:50; 16346‐1‐AP; Proteintech), TLR4 (1:400; GB11519; Servicebio), MMP‐3 (1:200; 17873‐1‐AP, Proteintech), MMP‐9 (1:400; GB11132; Servicebio), MMP‐13 (1:300; GB11247; Servicebio), IL‐1β (1:100; ab9722; Abcam), interleukin‐6 (IL‐6) (1:200; GB11117; Servicebio) and TNF‐α (1:100; ab6671; Abcam) overnight at 4°C in a humidified chamber. The sections were washed with phosphate‐buffered saline (PBS) and incubated with secondary antibodies. Finally, the sections were coloured by reacting with 3,3‐diaminobenzidine (DAB‐0031; Maixin). Haematoxylin was used for counterstaining the structure. Average optical density and positive cells count was determined by researchers who were blinded to the groups using ImageJ.
9
Renal Protein Expression Analysis
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Renal tissue proteins were extracted, quantified, and visualized using western blotting. First, target proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF, G6015-0.45, Servicebio) membranes. The PVDF membranes with bound target proteins were then blocked with 5% skim milk for 2 h. Primary antibodies (dilution 1 : 1000) against p-PI3K (BS-5570R, BIOSS, Beijing, China), PI3K (BSM-33219M, BIOSS), p-AKT (AF0908, Affinity, Changzhou, Jiangsu, China), AKT (3063, Cell Signaling Technology, Danvers, MA, USA), TLR4 (GB11519, Servicebio), and NF-κB (ab216409, Abcam, Cambridge, MA, USA) were used, and the membranes were incubated overnight at 4°C on a shaking bed. After washing the membranes, the secondary antibody (1 : 2,000 dilution) was added and incubated at 37°C for 2 h. After applying the ECL color reagent and performing dark chamber exposure imaging, the gray value of the images was analyzed using ImageJ software.
10
Immunohistochemical analysis of TLR4 and NF-kB
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Tissue biopsies were depara nized. Antigen retrieval was performed by EDTA, pH 8.0, for 23 min.
Posterior to natural cooling, the slides were put in PBS (PH = 7.4) and oscillated on a decolorising shaking device thrice, 5 min each. Sections were then preincubated with 10% normal goat serum for 30 min under 4°C to realize the blockade of non-speci c binding. Samples were afterwards cultivated for one night under 4°C with anti-TLR4 antibody (1:200; Servicebio, GB11519) or anti-NF-κB antibody (1:500, BS-0465R) in 2% serum; slices were cleaned thrice with PBS to realize the removal of unbound rst antibody and afterwards cultivated with second antibody for 60 min under 37°C. Subsequently, slices were cleaned ≥ 3 times with PBS involving 1% Tween 20 and covered with 4′,6-diamidino-2phenylindole/ uorescent quenching agent (Beyotime, PRC). Samples were observed via a Nikon uorescent microscope with a computerized camera (Nikon DS-U3) and three images per section were taken. TLR4 and NF-κB were analyzed by image-pro plus 6.0 (Media Cybernetics, MD, USA).
Posterior to natural cooling, the slides were put in PBS (PH = 7.4) and oscillated on a decolorising shaking device thrice, 5 min each. Sections were then preincubated with 10% normal goat serum for 30 min under 4°C to realize the blockade of non-speci c binding. Samples were afterwards cultivated for one night under 4°C with anti-TLR4 antibody (1:200; Servicebio, GB11519) or anti-NF-κB antibody (1:500, BS-0465R) in 2% serum; slices were cleaned thrice with PBS to realize the removal of unbound rst antibody and afterwards cultivated with second antibody for 60 min under 37°C. Subsequently, slices were cleaned ≥ 3 times with PBS involving 1% Tween 20 and covered with 4′,6-diamidino-2phenylindole/ uorescent quenching agent (Beyotime, PRC). Samples were observed via a Nikon uorescent microscope with a computerized camera (Nikon DS-U3) and three images per section were taken. TLR4 and NF-κB were analyzed by image-pro plus 6.0 (Media Cybernetics, MD, USA).
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