Each rat myocardial sample, formalin-fixed and paraffin-embedded, was cut into 5 μm sections. Slides were stained with haematoxylin and eosin (H&E) for standard histology. The distribution of collagen was carried out by Masson's trichrome staining and picrosirius red staining. The quantitative analysis of the cardiomyocyte size and collagen volume was measured with the Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA). Frozen sections were stained with Oil Red O to detect liquid content. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was utilized to assess cell death (Servicebio, Wuhan, China). After antigen retrieval in a citrate buffer (0.01 M, pH 6.0) and endogenous peroxidase blocking, slides were incubated with primary antibodies against collagen I (Affinity, AF0134, 1 : 100), collagen III (Affinity, AF0136, 1 : 100), NLRP3 (Abcam, ab210491, 1 : 200), ASC (Affinity, DF6304, 1 : 100), GSDMD (Affinity, AF4012, 1 : 100), IL-1β (Abcam, ab9722, 1 : 200), IL-18 (ProteinTech, 10663-1-AP, 1 : 300), and NEK7 (Affinity, DF4467, 1 : 100) at 4°C overnight. Thereafter, the tissues were incubated with HRP-conjugated secondary antibodies at 37°C for 30 minutes and observed with microscope (ZEISS, Axio Vert.A1, Jena, Germany).
Df6304
Manufactured by Affinity Biosciences
Sourced in United States
The DF6304 is a centrifuge designed for general laboratory use. It has a maximum speed of 6,000 rpm and a maximum relative centrifugal force of 3,900 xg. The centrifuge can accommodate a variety of rotor types and sample volumes.
Automatically generated - may contain errors
Lab products found in correlation
Af5103, by Affinity Biosciences (3 mentions)
Df7438, by Affinity Biosciences (3 mentions)
Et1610 93, by Huabio (2 mentions)
Et1608 69, by Huabio (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Af5418, by Affinity Biosciences (2 mentions)
Ab191860, by Abcam (2 mentions)
Ab9722, by Proteintech (1 mentions)
Af0134, by Affinity Biosciences (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Chemiluminescent kit, by Merck Group (1 mentions)
Af4012, by Affinity Biosciences (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Ultraview vox spinning disk confocal microscope, by PerkinElmer (1 mentions)
Ab109012, by Abcam (1 mentions)
Ab18256, by Abcam (1 mentions)
Fitc goat anti rabbit igg h l, by ABclonal (1 mentions)
Ab263899, by Abcam (1 mentions)
Ab179515, by Abcam (1 mentions)
8 protocols using df6304
1
Histological and Immunohistochemical Analysis of Rat Myocardium
2
Histopathological Analysis of TMJ Tissues
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TMJ tissues were harvested, fixed in 4% paraformaldehyde for 24 h, decalcified with EDTA (pH 7.4) for 2 weeks, and dehydrated and embedded in paraffin. Sliced into 4 μm thick, TMJ paraffin sections were prepared for hematoxylin and eosin (H&E) staining.
For immunohistochemical analysis, sections were deparaffinized and dehydrated with decreasing concentration ethanol. Repair antigens using 0.01 M citrate buffer at 95°C and cool down to room temperature. Slices were blocked with 3% H2O2 for 5 min to make endogenous peroxidase inactive and incubated overnight with primary antibodies including NLRP3 (1 : 100, ET1610-93, Huabio), ASC (1 : 100, DF6304, Affinity), GSDMD (1 : 100, AF4012), caspase-1 (1 : 100, ET1608-69, Huabio), Affinity), and IL-1β (1 : 100, AF5103, Affinity) at 4°C. Then, the sections were washed with PBS and incubated with HRP-conjugated secondary antibodies for 15 min and DAB for 5 min. The immunoreaction was observed by a Zeiss microscope (Carl Zeiss MicroImaging) and quantified by using ImageJ software (NIH).
For immunohistochemical analysis, sections were deparaffinized and dehydrated with decreasing concentration ethanol. Repair antigens using 0.01 M citrate buffer at 95°C and cool down to room temperature. Slices were blocked with 3% H2O2 for 5 min to make endogenous peroxidase inactive and incubated overnight with primary antibodies including NLRP3 (1 : 100, ET1610-93, Huabio), ASC (1 : 100, DF6304, Affinity), GSDMD (1 : 100, AF4012), caspase-1 (1 : 100, ET1608-69, Huabio), Affinity), and IL-1β (1 : 100, AF5103, Affinity) at 4°C. Then, the sections were washed with PBS and incubated with HRP-conjugated secondary antibodies for 15 min and DAB for 5 min. The immunoreaction was observed by a Zeiss microscope (Carl Zeiss MicroImaging) and quantified by using ImageJ software (NIH).
3
Western Blot Analysis of NLRP3 Inflammasome
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The protein from tissue or cultured cell was extracted using RIPA buffer (R0010, Solarbio, China). Lysates were obtained by centrifugation at 4°C with 12000 rpm for 15 min. Protein levels were quantified with a BCA protein assay kit (Beyotime Biotechnology, China). Then, total protein was separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Lot#K5NA8025F, Millipore, USA). Block membranes with 5% skim milk TBS-T for 1.5 h. Incubate them overnight at 4°C with primary antibodies against NLRP3 (1 : 1000, ET1610-93, Huabio), ASC (1 : 1000, DF6304, Affinity), caspase-1 (1 : 1000, ET1608-69, Huabio), IL-1β (1 : 1000, AF5103, Affinity), GSDMD (1 : 1000, AF4012, Affinity), and GAPDH (1 : 1000, 181602, Abcam). The membranes were then washed with TBS-T, followed by incubation with secondary antibodies at room temperature for 1.5 h. After washing 3 times with TBS-T, blot signals were visualized by chemiluminescent kit (Millipore, USA).
4
Immunofluorescence Analysis of Inflammasome Proteins
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The cells were plated into 12-well plates and fixed with 3% formaldehyde (Sinopharm, China) for 15 min. After washing three times with PBS, the cells were permeabilized with Triton-X 100 (1%) for 10 min. Subsequently, cells were blocked with bovine serum albumin (3%) for 30 min. Next, cells were labeled with primary antibodies including anti-caspase-1 (1:500; AF5418, Affinity, USA), anti-ASC (1:100; DF6304, Affinity), anti-DDX3X (1:50; ab271002, Abcam, UK), anti-SQSTM1 (1 μg/mL; ab109012, Abcam), anti-HMGB1 (1 μg/mL; ab18256, Abcam), and anti-NLRP3 (1:100; ab263899, Abcam) overnight at 4° C. Following that, cells were incubated with the secondary antibody FITC goat anti-rabbit IgG (H+L) (ABclonal, MA, USA) and 4’,6-diamidino-2-phenylindole (DAPI, 1:500) for 1 h in the dark. Finally, the cells were visualized using an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer, MA, USA).
5
Neuroinflammatory Markers in Spinal Cord Injury
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Spinal cord slices (4-μm-thick) from six rats in each group were generated on the 22nd day after surgery, as described in the Histological analysis subsection. The sections were treated with 3% H2O2 solution in the dark for 25 minutes to block endogenous peroxidase. After rinsing in phosphate-buffered saline, the sections were sealed with 5% bovine serum albumin (37°C, 30 minutes) followed by incubation with an anti-apoptosis-associated speck-like protein containing CARD (ASC) rabbit polyclonal antibody (1:200, Affinity, Cat# DF6304, RRID: AB_2838270), anti-caspase-1 rabbit polyclonal antibody (1:200, Affinity, Cat# AF5418, RRID: AB_2837902), anti-NLRP3 rabbit polyclonal antibody (1:200, Affinity, Cat# DF7438, RRID: AB_2839376), and anti-Nrf2 rabbit polyclonal antibody (1:200, Affinity, Cat# AF0639, RRID: AB_2833793) overnight at 4°C. Afterwards, goat anti-rabbit IgG (H+L) HRP (1:200, Affinity, Cat# S0001, RRID: AB_2839429) was added (37°C, 1 hour), the nuclei were counterstained with DAPI, and the slices were dehydrated and mounted with neutral balsam. An optical microscope was used to visualize the tissues, and Image Pro Plus 6.0 was used to determine the integrated optical density (IOD) and average density (AOD) = IOD/area. AOD was used to analyze immunopositivity.
6
Western Blot Analysis of Inflammatory Markers
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The cells were lysed using lysis buffer (P0013B; Beyotime, Shanghai, China) containing PMSF (329-98-6; Nanjing Wohong, Nanjing, China) and lysate. Protein concentration was quantified using a BCA Protein Assay kit (P0009; Beyotime). Cell lysates were separated by 10% SDS-PAGE and transferred to an NC membrane (Sigma-Aldrich, St Louis, USA). After being blocked with 5% skim milk for 2 h, the membrane was incubated with the following primary antibodies: fibrinogen antibody (ab281924, 1:1000; Abcam), matrix metalloprotein 9 (MMP9) antibody (ab283575, 1:1000; Abcam), ZO-1 antibody (ab61357, 1:1000; Abcam), occludin antibody (ab216327, 1:1000; Abcam), pro caspase1 (ab179515, 1:1000; Abcam), cleaved caspase1 (AF4005, 1:1000; Affinity), NLRP3 (DF7438, 1:1000; Affinity), ASC (DF6304, 1:1000; Affinity), IL-1β (ab234437, 1:100; Abcam), IL-18 (ab191860, 1:100; Abcam), p65 antibody (ab32536, 1:2000; Abcam), p-p65 antibody (Ma5-15160, 1:1000; Thermo Fisher Scientific, Waltham, USA), GAPDH antibody (ab8245, 1:2000; Abcam). The membrane was then incubated for 1 h with HRP-labeled secondary antibodies (ab6721, 1:3000; Abcam). After three times wash with PBS, the membrane was detected using BeyoECL Plus (Beyotime).
7
Protein Expression and NLRP3 Inflammasome
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The protein concentration was calculated by using bicinchoninic acid (BCA) assay kit (KGI Biotechnology Co. Ltd. China). Identical quantities of protein samples were denatured by protein loading buffer, separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, then transferred to PVDF membranes (Millipore, Billerica, MA, United States). The PVDF membranes were sealed with 5% bovine serum albumin (BSA) at room temperature for one hour. The following antibodies were incubated at 4°C overnight: anti-CAMK4 (1:1,000, A5304, ABclonal), anti-p-CREB (1:1,000, ab119711, Abcam), anti-CREB1 (1:1,000, A0011, ABclonal), anti-NLRP3 (1:500, DF7438, Affinity Biosciences), anti-ASC (1:500, DF6304, Affinity Biosciences), anti-CASP1 (1:500, AF5418, Affinity Biosciences). The stained blots were detected using an ECL reagent (Thermo Fisher Scientific, MA, United States). Each blot was quantified using Bio-Rad Image Lab™ Version 3.0 software. All the experiments reported in this study were performed three times, and the results were reproducible.
8
Selenious Yeast Tablet Therapeutic Effects
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Selenious Yeast Tablet (Selenious) was obtained from Mudanjiang Lingtai Pharmaceuticals CO., Ltd. (Mudanjiang, China). Porcine thyroglobulin (pTg), complete Freund’s adjuvant (CFA) and incomplete Freund’s adjuvant (IFA) were provided by Sigma Aldrich (CA, United States). Lugol’s iodine solution was supplied by Preparation Room of Jiangsu Traditional Chinese and Western Medicine Hospital (Nanjing, China). The antibodies were purchased from CST (Danvers, United States) and Abcam (Burlingame, United States). Antibodies to Wnt-1 (AF5315, Affinity), β-catenin (8,480, CST), IL-17A (DF6127, Affinity), p-NF-κB p65 (3,039, CST), NF-κB p65 (8,242, CST), NLRP3 (ab214185, Abcam), ASC (DF6304, Affinity), Cleaved-Caspase1 (AF4022, Affinity), Cleaved-IL-1β (AF5103, Affinity), IL-18 (ab191860, Abcam), β-Tubulin (AF7011, Affinity) and HRP-conjugated secondary antibodies (7,074, CST) were used for western blot. NLRP3 (DF7438, Affinity) and Alexa Fluor® 488 secondary antibodies (62,304, CST) were used for the immunofluorescent detection of NLRP3.
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