For analysis of IL-1β and TNF-α expression, the tissue sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity then incubated again with 3% bovine serum albumin in PBS to reduce the non-specific background. After incubation with primary antibodies against IL-1β (1:200; MAB601, R&D systems, Minneapolis, MN, USA) or TNF-α (1:200; A11534, ABclonal, Woburn, MA, USA) at 4 °C overnight, followed by incubation with secondary antibody (1:200) at room temperature for 1 h, the sections were stained with diaminobenzidine and observed under a light microscope, as previously described [35 (link),36 (link),37 (link)]. The intensities of the immunoreactive signals were scored from 1 to 5 (from weak to strong) for positive expression by two independent assessors who were blinded to the treatment groups to minimize observer bias.
A11534
Manufactured by ABclonal
Sourced in China
A11534 is a lab equipment designed for general laboratory use. It serves as a tool to assist in various experimental procedures. The core function of this product is to provide a reliable and versatile solution for laboratory tasks, without making any claims about its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Mab601, by R&D Systems (1 mentions)
A5786, by ABclonal (1 mentions)
αsma a2547, by Merck Group (1 mentions)
Sc 1265, by Santa Cruz Biotechnology (1 mentions)
Collagen 1, by ABclonal (1 mentions)
As014, by ABclonal (1 mentions)
Wl01114, by Wanlei (1 mentions)
A1112, by ABclonal (1 mentions)
A 21050, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Phosphate buffer saline (pbs), by Merck Group (1 mentions)
Ab208113, by Abcam (1 mentions)
Imagej, by LI COR (1 mentions)
Sc 271185, by Santa Cruz Biotechnology (1 mentions)
Hpa001239, by Merck Group (1 mentions)
7 protocols using a11534
1
Quantifying IL-1β and TNF-α Expression
2
Immunohistochemical Analysis of Tissue Samples
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The tissue sections were fixed with acetone for 5 min and air-dried for 10 min. The sections were then immersed in 0.3% H2O2/PBS for 5 min to remove peroxidase activity. After treating with blocking solution for 1 h, primary antibodies were applied and incubated for 1 h, followed by secondary antibodies (HRP-conjugated goat anti-rabbit/mouse IgG), reacting for another hour. The sections were then stained with DAB for 5 min and rinsed with distilled water for another 10 min. The primary antibodies used in this experiment were TGF-β (sc-146, Santa Cruz), α-SMA (A2547, Sigma-Aldrich), Collagen I (A5786, ABclonal), IL-6 (sc-1265, Santa Cruz), IL-17A (A12454, ABclonal), and TNF-α (A11534, ABclonal).
3
IHC Staining of Inflammatory Markers
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Immunohistochemical (IHC) staining of TNF-α, IL-6 and IL-1β expression in lung tissues was performed as previously described (Shi et al., 2022 (link)). Briefly, lung tissues were fixed in 4% paraformaldehyde solution for 48 h before incubation with primary antibodies for the detection of TNF-α (ABclonal Technology Co., Ltd., Wuhan, China, A11534) and IL-6 (ABclonal, A21264) and IL-1β (Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China), K108840P). The secondary antibody was purchased from Servicebio Technology Co., Ltd., (Wuhan, China).
4
Regulation of Macrophage Inflammation
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The materials used in this study are as follows: mouse mononuclear macrophage leukemia cell line (Raw264.7, Chinese Academy of Sciences, China), Roswell Park Memorial Institute (RPMI) 1640 cell culture medium (Thermo Fisher, USA), HDAC9-shRNA and NC-shRNA (Obio Technology Corporation, Ltd. Shanghai, China), phosphate buffer saline pH 7.4 (Sigma Aldrich, USA), Triton X-100 (T8200, Soleibo, China), TLR4 inhibitor (B4935, APExBio Shanghai, China), HDAC9 (PA5–11245, 1 : 1000) and TLR4 (MAI-33380, 1 : 1000) (Invitrogen, USA, 1 : 1000), IL-1β (A1112, ABclonal, China, 1 : 500), CD38 (A20215, ABclonal, China, 1 : 500), TNF-α (A11534, ABclonal, China, 1 : 500), GAPDH (WL01114, Wanleibio, China, 1 : 10000), HRP goat anti-rabbit IgG (AS014, ABclonal, China, 1 : 10000), and fluorescent secondary antibodies A-11008 and A-21050 (Invitrogen, USA). All chemicals were used without any further processing.
5
Quantifying Neuroinflammatory Markers in Skull Sections
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Immunohistochemical staining was used to detect the expression of IL-1β(1:100, #12242, CST),IL-6 (1:50, ab208113, abcam) and TNF-α(1:100, A11534, ABclonal). Skulls sectioned were incubated with the respective primary antibodies overnight at 4 °C. Sections were then washed and incubated with secondary antibody for 30 min at room temperature. Stained sections were all photographed using light microscope. Histomorphological analysis of bones was performed via Panorama Histocytometry Quantitative Analysis System (TissueFAXS Plus, TissueGnostics GmbH, Austria).
6
Western Blot Analysis of Cardiac Proteins
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Total protein was extracted from fresh tissues and cells based on the previous protocol (Song et al., 2021 (link)). Briefly, 50 μg protein samples were separated in 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. After 5% non-fat milk blocking, the blots were incubated with primary antibodies including BNP (1:200 dilution, sc-271185, Santa Cruz), β-MHC (1:500 dilution, HPA001239, Sigma), PI3K (1:2,000 dilution, A12484, Abclonal), LC3-II (1:2,000 dilution, A11534, Abclonal), Akt (1:2,000 dilution, A16343, Abclonal), and β-actin (1:5,000 dilution, KC5A08, Kangcheng) at 4°C overnight. Imaging System (LI-COR Biosciences) and ImageJ were used to perform a statistical analysis.
7
Immunohistochemical Profiling of Gastric Tumor Markers
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Thirty paraffin-embedded tissue slides were subjected to immunohistochemical staining using the following antibodies: PD-L1 (AG27557, Proteintech), PD-1 (AG12470, Proteintech), p65 (AG1199, Proteintech), TRAF6 (AG24296, Proteintech), CagA (A-10, Santa Cruz Biotechnology), tumor necrosis factor-α (TNF-α) (A11534, ABclonal), interferon (IFN)-γ (AG8321, Proteintech), and IL-10 (AG14870, Proteintech). Staining of preprocessed endoscopic biopsy samples and surgical specimens was carried out on embedded slices using the indirect peroxidase method (pH 6.0 in sodium citrate buffer). Two expert pathologists scored the slides independently, bypassing the clinical data. An IScope microscope (IS.1159EPLi, Euromex) and Image Focus V.4.0 (Euromex) software were applied to scan and capture the images. The H-score to quantify the P65, TRAF6, TNF-α, IFN-γ, IL-10, PD-1, and PD-L1 staining intensities was calculated as follows: ∑ (3×percentage of strong staining +2× percentage of moderate staining +percentage of weak staining) using ImageJ with 0–300 fluctuations. The graphic data were displayed as mean±SE of the mean (SEM). For CagA markers, the positive expression exceeded 10% in cells with moderate or strong immunostaining.15 (link)
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