Western blotting was performed using published protocols [2 (link),3 (link),33 (link)] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).
Df6894
Manufactured by Affinity Biosciences
Sourced in United States
The DF6894 is a laboratory centrifuge designed for general-purpose applications. It is capable of separating samples based on density differences. The centrifuge features a rotor with a maximum speed of 6,000 RPM and a maximum relative centrifugal force (RCF) of 4,000 x g.
Automatically generated - may contain errors
Lab products found in correlation
Af5103, by Affinity Biosciences (4 mentions)
Df6087, by Affinity Biosciences (3 mentions)
S0001, by Affinity Biosciences (2 mentions)
Af5142, by Affinity Biosciences (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Df6280, by Affinity Biosciences (1 mentions)
Arg56509, by Arigo Biolaboratories (1 mentions)
Gtx112141, by GeneTex (1 mentions)
Ac026, by ABclonal (1 mentions)
Gtx110520, by GeneTex (1 mentions)
A7277, by ABclonal (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Kit 9706, by Maixin Group (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Sirius red, by Merck Group (1 mentions)
Af1027, by Affinity Biosciences (1 mentions)
Ab188570, by Abcam (1 mentions)
Chemiluminescent reagent, by Beyotime (1 mentions)
Df7561, by Affinity Biosciences (1 mentions)
Ap0489, by ABclonal (1 mentions)
6 protocols using df6894
1
Western Blot Analysis of Metabolic and Immune Markers
2
Immunohistochemical and Collagen Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were harvested and fixed with 10% formalin and embedded in paraffin. Serial sections (4 μm) were prepared and stained with H&E and immunohistochemistry (IHC). For IHC, tissue sections were subjected to antigen retrieval and incubation with primary antibody against CCL2 (BF0556, Affinity), TGF-β (AF1027, Affinity), IL-1β (AF5103, Affinity), IL-6 (DF6087, Affinity), or IL-10 (DF6894, Affinity), followed by incubation with a peroxidase-conjugated goat anti-rabbit IgG (KIT-9706, Maixin-Bio) secondary antibody, and the immunoreactivity was detected with an UltraSensitive™ Streptavidin-Peroxidase Kit (KIT-9710, Mai Xin, China) according to the manufacturer's protocol. The collagen content was assessed by Sirius Red (365548, Sigma). For immunofluorescence (IF), cryosections (4 μm) were prepared from freshly frozen liver tissues and incubated with antibodies specific for mouse CD68 (137005, Biolegend), CD31-antibody (102432, Biolegend), TIE2 (124008, Biolegend), or phos-NF-κB (Affinity, AF5006). Photomicrographs were produced using a confocal microscope and corresponding software (Zeiss). Image Pro Plus 6.0 software was used for quantitative analysis.
3
Mechanotransduction and Anti-inflammatory Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed to assess the influence of the
CAV1-YAP-mediated mechanotransduction pathway and the anti-inflammatory
effects of the NPC-encapsulated composite hydrogel under inflammatory
conditions. Proteins were labeled and incubated overnight at 4°C with the
following primary antibodies: YAP (1:500, A1001, ABclonal), p-YAP (1:500,
AP0489, ABclonal), CAV1 (1:2000, 16447-1-AP, Proteintech), Col II (1:2000,
ab188570, Abcam), Agg (1:2000, DF7561, Affinity), IL-1β (1:1000, AF5103,
Affinity), IL-6 (1:1000, DF6087, Affinity), IL-10 (1:1000, DF6894,
Affinity), IL-4 (1:1000, AF5142, Affinity), and β-actin (1:5000, AF7018,
Affinity). After washing using TBST buffer, the membranes were treated with
horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, S0001,
Affinity) for 2 h at room temperature. Then examined with a chemiluminescent
reagent (Beyotime), and quantified the signal intensity using ImageJ
software.
CAV1-YAP-mediated mechanotransduction pathway and the anti-inflammatory
effects of the NPC-encapsulated composite hydrogel under inflammatory
conditions. Proteins were labeled and incubated overnight at 4°C with the
following primary antibodies: YAP (1:500, A1001, ABclonal), p-YAP (1:500,
AP0489, ABclonal), CAV1 (1:2000, 16447-1-AP, Proteintech), Col II (1:2000,
ab188570, Abcam), Agg (1:2000, DF7561, Affinity), IL-1β (1:1000, AF5103,
Affinity), IL-6 (1:1000, DF6087, Affinity), IL-10 (1:1000, DF6894,
Affinity), IL-4 (1:1000, AF5142, Affinity), and β-actin (1:5000, AF7018,
Affinity). After washing using TBST buffer, the membranes were treated with
horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, S0001,
Affinity) for 2 h at room temperature. Then examined with a chemiluminescent
reagent (Beyotime), and quantified the signal intensity using ImageJ
software.
4
Evaluating ECM, Inflammatory Markers, and Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate in vivo changes in ECM secretion, levels of
inflammatory cytokines, and macrophage polarization, the sections were
immunohistochemically stained. Briefly, deparaffinized sections were
incubated with 3% H2O2 for 15 min in the dark and then
blocked with 3% BSA for 30 min at room temperature. The sections were
incubated overnight at 4°C along with the following primary antibodies: Col
II (1:500, GB111629, Servicebio), aggrecan (1:500, GB11373, Servicebio),
IL-1β (1:200, AF5103, Affinity), IL-6 (1:200, DF6087, Affinity), IL-10
(1:200, DF6894, Affinity), IL-4 (1:200, AF5142, Affinity), CD68 (1:300,
GB113109, Servicebio), Arg1 (1:500, GB11285, Servicebio), and CC-chemokine
receptor 7 (CCR7) (1:300, GB11502, Servicebio). After primary antibody
incubation, the sections were incubated with a specific HRP-conjugated
secondary antibody (1:200, S0001, Affinity) for 1 h at 25°C. The sections
were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin
for the nucleus. Finally, the sections were mounted and photographed using
an optical microscope (Nikon, Tokyo, Japan). Immunohistochemical staining
intensity was digitally quantified using ImageJ software.
inflammatory cytokines, and macrophage polarization, the sections were
immunohistochemically stained. Briefly, deparaffinized sections were
incubated with 3% H2O2 for 15 min in the dark and then
blocked with 3% BSA for 30 min at room temperature. The sections were
incubated overnight at 4°C along with the following primary antibodies: Col
II (1:500, GB111629, Servicebio), aggrecan (1:500, GB11373, Servicebio),
IL-1β (1:200, AF5103, Affinity), IL-6 (1:200, DF6087, Affinity), IL-10
(1:200, DF6894, Affinity), IL-4 (1:200, AF5142, Affinity), CD68 (1:300,
GB113109, Servicebio), Arg1 (1:500, GB11285, Servicebio), and CC-chemokine
receptor 7 (CCR7) (1:300, GB11502, Servicebio). After primary antibody
incubation, the sections were incubated with a specific HRP-conjugated
secondary antibody (1:200, S0001, Affinity) for 1 h at 25°C. The sections
were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin
for the nucleus. Finally, the sections were mounted and photographed using
an optical microscope (Nikon, Tokyo, Japan). Immunohistochemical staining
intensity was digitally quantified using ImageJ software.
5
Hippocampal Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed following the protocol from previous studies (Fan Y. et al., 2015 (link)). The anesthetized rats were transcardially perfused with saline. The brains were removed, and the hippocampi were dissected. Lysis buffer was prepared by RIPA and PMSF according to a ratio of 100:1, and one tablet phosphatase inhibitor (4906837001, Roche, Switzerland) was added to every 10 mL lysis buffer. The tissues were homogenized in lysis buffer (containing phosphatase inhibitor). After cracking and centrifugation, the supernatant was collected. Equal amounts of total proteins were resolved by SDS-PAGE and transferred on to a nitrocellulose membrane. The membranes were incubated overnight at 4°C with the following primary antibodies: anti-STAT6 (ab217998, 1:1,000, Abcam, USA), anti-pSTAT6 (ab263947, 1:1,000, Abcam, USA), anti-TGF-β1 (ab92486, 1:1,000, Abcam, USA), anti-IL-10 (DF6894, 1:500; Affinity, USA), anti-IL-1β (AF5103, 1:500; Affinity, USA), anti-IFN-γ (ab9657, 1:1,000, Abcam, USA), and anti-α-tubulin (2144, 1:1,000, CST, USA). Then, the membranes were washed with 1 × TBST (10 mM Tris-HCl+10 mM NaCl+0.05%Tween-20), incubated with appropriate secondary antibodies: HRP-conjugated anti-rabbit antibody (5571, 1:3,000, CST, USA), at room temperature for 1 h, and then treated with ECL substrate (Millipore, USA).
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from tissues or cells using RIPA lysis buffer (CW2333, CWBIO, China) containing proteinase and phosphatase inhibitors. Nuclear and cytoplasmic protein of TM3 cells was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Western blot analysis was conducted as previously described [35 (link)]. The primary antibodies included anti-FOXO4 (ab128908, 1:1000; Abcam, USA), anti-p53 (ab26, 1:200; Abcam, USA), anti- Ser15-phospho-p53 (ab1431, 1:500; Abcam, USA), anti-p21 (ab188224, 1:1000; Abcam, USA), anti-p16 (MAB2416, 1:500; Abnova, USA), anti-3β-HSD (sc-515120, 1:200; Santa Cruz, USA), anti-CYP11A1 (GTX56293, 1:500; Gene Tex, USA), anti-CYP17A1 (ab125022, 1:1000; Abcam, USA), anti-IL-1α (16764-1-AP, 1:500; Proteintech, USA), anti-IL-1β (ab9722, 0.2 μg/ml; Abcam, USA), anti-IL-6 (ab9324, 0.4 μg/ml; Abcam, USA), anti-IL-10 (DF6894, 1:500; Affinity, USA), anti-TNF-α (17590-1-AP, 1:500; Proteintech, USA), anti-TGF-β (18978-1-AP, 1:500; Proteintech, USA) and anti-GAPDH (T0004, 1:5000; Affinity, USA) antibodies. GAPDH was used as the control.
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!