Paraffin-embedded tissue sections (5 μm) were deparaffinized and hydrated with xylene and graded alcohols using a standard protocol, and incubated overnight with primary antibodies at 4 °C in a humidified chamber, and then rinsed and incubated with biotinylated secondary antibodies for 30 min at room temperature. The slides were developed using the ImmunoCruz™ ABC Staining System (Santa Cruz Biotechnology) and were counterstained with Mayer’s hematoxylin solution (Wako). Immunohistochemical staining with anti-cadherin-11 (Abcam, # ab151302), anti-CD11b (Abcam, #ab133357), and anti-CD3 (Abcam, #ab16669) antibodies was performed. Assessment of the extents of cell populations were performed by using semi-quantitative assessment (SQA) reported previously36 (link)–38 (link). In summary, SQA was performed in three high power fields (HPFs; ×400) in each sample by two independent observers. The expression of cadherin-11, CD11b, and CD3 was scored on a 5-point scale (0–4; score 0 represented minimal infiltration, while score 4 represented infiltration by numerous cells) and the mean of the three scores was calculated in each sample.
Ab151302
Manufactured by Abcam
Sourced in United Kingdom
Ab151302 is a polyclonal antibody that targets the SERPINH1 protein. It is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab34710, by Abcam (2 mentions)
Ab33902, by Abcam (2 mentions)
Ab227518, by Abcam (2 mentions)
Ab13418, by Abcam (2 mentions)
Ab192256, by Abcam (2 mentions)
Ab73211, by Abcam (2 mentions)
Ab52917, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Mayer s hematoxylin solution, by Fujifilm (1 mentions)
Ab16669, by Abcam (1 mentions)
Ab133357, by Abcam (1 mentions)
Immunocruz abc staining system, by Santa Cruz Biotechnology (1 mentions)
Dab substrate chromogen system, by Agilent Technologies (1 mentions)
Cy3 conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Fitc conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Ab32572, by Abcam (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Nf κb, by Abcam (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
7 protocols using ab151302
1
Immunohistochemical Analysis of Cell Populations
2
Immunohistochemical Analysis of CDH11 Expression
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Immunohistochemistry was performed using the diaminobenzidine chromogen method as described previously.13 Briefly, paraffin‐embedded tissues were deparaffinized by using xylol and descending concentrations of ethanol. Then, 0.3% H2O2 was used to block the endogenous peroxidases. After antigen retrieval with Citrate buffer, blocking was performed by using 5% bovine serum albumin (BSA). Then, incubation with primary antibodies against CDH11 (ab151302; Abcam, Cambridge, MA) was performed. The slides were rinsed in phosphate‐buffered saline (PBS), and the slides were incubated with the secondary antibody (HRP conjugated goat antirabbit IgG, GB23303; Servicebio) for 1 h at RT. Antibody binding was visualized by using a liquid DAB Substrate Chromogen System (Dako, Glostrup, Denmark). The slides were rinsed in PBS and then the slides were counterstained with hematoxylin.
3
Immunofluorescence Assay for Liver Tissue Analysis
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The immunofluorescence assays were performed using the methods described previously.13 , 22 Briefly, the liver sections were incubated with xylol and descending concentrations of ethanol. The endogenous peroxidases were removed by using 0.3% H2O2. After blocking with 5% BSA, the CDH11, alpha‐smooth muscle actin (α‐SMA), and phosphorylated small mothers against decapentaplegic homolog 2 (p‐Smad2) antibodies (CDH11, ab151302; Abcam, α‐SMA, ab7817; Cambridge, MA, p‐Smad2, 18338; Cell Signaling Technology, Danvers, MA) was applied overnight in a wet chamber. The secondary antibodies were applied to the slides for 1 h after washing three times with PBS at RT. The FITC conjugated goat antirabbit IgG (GB22303; Servicebio), Cy3 conjugated goat antimouse IgG (GB21301; Servicebio), and Cy3 conjugated goat antirabbit IgG (GB21303; Servicebio) were used as secondary antibodies. Then, the nuclei were counterstained with 4′, 6‐diamidino‐2‐phenylindole (DAPI). The positive staining signals were detected by fluorescent microscopy (Nikon, Tokyo, Japan).
4
Comprehensive Protein Expression Analysis
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Total protein was extracted by RIPA buffer, and protein concentration was detected using a BCA protein quantification kit. A 30 μg protein sample was used for 10% SDS-PAGE electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane, and the membrane was blocked with 5% BSA. Primary antibody was added to the membrane and incubated overnight, and then incubated with an HRP-labeled secondary antibody, and ECL development was performed after the incubation. The antibodies were as follows: COL1A1 (Abcam, #ab34710)9 (link), RUNX2 (Abcam, #ab192256)10 (link), OCN (Abcam, #ab13418)11 (link), p-SMAD1 (Abcam, #ab73211)12 (link), t-SMAD1 (Abcam, #ab33902)13 (link), CDH11 (Abcam, #ab151302)14 (link), VEGF (Abcam, #ab52917)15 , beta-catenin (Abcam, #ab32572)16 (link), Dicer (Abcam, #ab227518), and GAPDH (Abcam, #ab181602)17 (link).
5
Western Blot Analysis of Chicken Myoblasts
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Chicken primary myoblast cellular proteins were extracted by using radio-immunoprecipitation assay (RIPA) buffer and phenylmethanesulfonyl fluoride (PMSF) protease inhibitor. Western blot assays were performed as previously reported (Bass et al., 2017 (link)). The antibodies used were as follows: rabbit anti-OB Cadherin (ab151302; Abcam, United States; 1:1000), goat anti-rabbit IgG-HRP (BA1055; Boster, China; 1:2500), rabbit anti-GAPDH (AB-P-R 001; Goodhere, China; 1:1500).
6
Western Blot Analysis of Apoptosis Markers
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A total of 40 mg of protein lysate was subjected to western blotting as previously described.16 (link) The primary antibodies used were as follows: CDH11 (1:1,000, ab151302; Abcam, Cambridge, UK), cleaved-caspase 3 (1:500, WL03089; Wanleibio, Shenyang, China), cleaved-caspase 9 (1:500, WL01838; Wanleibio, Shenyang, China), cleaved-PARP (1:1,000, 7851; Cell Signaling Technology), BAX (5023; Cell Signaling Technology), NF-κB (ab16502; Abcam, Cambridge, UK), phospho-NF-κB (3031s; Cell Signaling Technology), Bcl-2 (2872T; Cell Signaling Technology), Bcl-XL (2762S; Cell Signaling Technology), and β-actin (LK-ab008-100; Liankebio, China) was used as control. Anti-mouse IgG (7076, 1:3,000 Cell Signaling Technology) and anti-rabbit IgG (7074, 1:2,000 Cell Signaling Technology) horseradish peroxidase conjugate secondary antibodies were used. Protein blots were analysed using an enhanced chemiluminescence detection kit (ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA), and the membranes were visualized with a Las-4,000 Imaging System (Medical Systems, Fujifilm Global, Tokyo, Japan).
7
Protein Expression Analysis of Osteogenic Markers
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Cells were lysed on ice in RIPA protein lysate solution for 30 minutes. The cell debris was pelleted by centrifugation for 10 min at 10,000 rpm. Protein concentrations in the supernatant were determined using the bicinchoninic acid (BCA) method. Samples in sample buffer were heated for 10 min at 98° C. Samples containing 20 μg protein were resolved by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels using a vertical transfer system (#1658033 Bio-Rad Laboratories Inc.). The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Nonspecific protein binding on the membranes was blocked with 5% BSA for 1 h and the membranes were incubated overnight at 4° C with the primary antibodies. The membranes were then washed with TBST for 1 h, incubated with HRP-labeled secondary antibodies, and finally washed with TBST. Bound proteins on the blots were visualized using an automatic chemiluminescence imaging analysis system (#5200; Tanon Science and Technology Co., Ltd., Shanghai, China). The primary antibodies specific to COL1A1 (#ab34710), RUNX2 (#ab192256), OCN (#ab13418), p-SMAD1 (#ab73211), t-SMAD1 (#ab33902), RUNX2 (#ab151302), VEGF (#ab52917), beta-catenin (#ab32572), Dicer (#ab227518), and GAPDH (#ab181602) were purchased from Abcam (Cambridge, UK).
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