Lung tissue from mice was ground into homogenates at the condition of 4°C for protein detection [26 (link)]. Total protein was extracted via a Total Protein Extraction Kit (Invent Biotechnologies Inc., USA) according to the manufacturer's protocol. Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China) was used for concentration detection. Protein samples were then separated by SDS-PAGE gel electrophoresis on 12% glycine-based gels and transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked in 5% (w/v) skimmed milk for 2 h and incubated with antibodies in 5% (w/v) BSA overnight at 4°C. Anti-Foxp3 antibody, anti-RORγt antibody, and anti-GAPDH antibody were purchased from Abcam (United Kingdom). Membranes were then incubated with secondary HRP-labeled antibody (Sigma-Aldrich, St. Louis, MO, USA) in 5% (w/v) BSA for 1 h. Afterwards, enhanced chemiluminescence (Beyotime, Jiangsu, China) was developed on membranes. The intensity of bands was quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Anti foxp3 antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-FOXP3 antibody is a protein that binds to and detects the FOXP3 transcription factor. FOXP3 is a key regulator of the development and function of regulatory T cells. The antibody can be used to identify and quantify FOXP3-positive cells in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Hrp labeled antibody, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Total protein extraction kit, by Invent Biotechnologies (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Ab150686, by Abcam (1 mentions)
Anti cd4 ab, by Leica camera (1 mentions)
Protease inhibitor cocktail, by MedChemExpress (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Anti gapdh antibody, by Bioworld Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Hrp dab abc detection ihc kit, by Abcam (1 mentions)
Anti mcd8, by Thermo Fisher Scientific (1 mentions)
Anti mcd4, by Abcam (1 mentions)
Immpress horse anti rabbit igg polymer kit, by Vector Laboratories (1 mentions)
Vector sg peroxidase hrp substrate kit, by Vector Laboratories (1 mentions)
Anti cd3 antibody, by Abcam (1 mentions)
9 protocols using anti foxp3 antibody
1
Quantification of Lung Protein Markers
2
Immunohistochemical Analysis of IgG4, CD38, and Foxp3
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Paraffin sections (4 μm) were stained with H&E, periodic acid Schiff stain or periodic acid-methenamine silver. For immunohistochemical staining, kidneys were snap-frozen in optimal cutting temperature compound (Sakura Finetek Japan Co. Ltd, Tokyo, Japan). The sections were immunostained using mouse monoclonal antibodies against human IgG4 (Zymed Laboratory, San Francisco, CA, USA, or The Binding Site, Birmingham, UK), anti-CD38 antibody (Dako, Glostrup, Denmark) and anti-Foxp3 antibody (Abcam, Cambridge, UK).
3
FOXP3 Regulation of NGFR Promoter
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The interaction between FOXP3 and NGFR promoter was determined by ChIP as previously described (12 (link)). Briefly, anti-FOXP3 antibody (Abcam) was used for immunoprecipitating the DNA fragments that interacted with FOXP3, followed by PCR and agarose gel electrophoresis. The PCR of the NGFR promoter sequence was formed using 40 cycles as follows: 95 °C for 15 seconds, 65 °C for 40 seconds. The PCR products were analyzed on 1.5% agarose gels.
4
Evaluation of Renal Immune Cells
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Formalin fixation and paraffin embedding of the bilateral renal specimens were performed according to previously described procedures [16 (link)]. The primary antibodies used were anti-mouse mAbs (anti-CD4 Ab, Leica, Leica Biosystems, Newcastle, UK and anti-IL-17 Ab, Abcam, Cambridge, UK), and the secondary antibody used was a rabbit monoclonal anti-FOXP3 antibody (Abcam, Cambridge, UK). Renal fibrosis was assessed with Masson trichrome staining using a commercial kit (Abcam; ab150686).
The semiquantitative evaluations and grading of the IHC staining were performed by two independent investigators. The number of CD4+FOXP3+, CD4+IL-17+, or FOXP3+IL-17+ cells was determined according to the number of positive cells per high-powered field (HPF) at 400X magnification from 10 randomly chosen fields within the same section of kidney tissue from an individual biopsy specimen [14 (link)]. The interstitial volume, α-smooth muscle actin (α-SMA) score were calculated by the point counting method in accordance with previous reports [16 (link), 18 (link)].
The semiquantitative evaluations and grading of the IHC staining were performed by two independent investigators. The number of CD4+FOXP3+, CD4+IL-17+, or FOXP3+IL-17+ cells was determined according to the number of positive cells per high-powered field (HPF) at 400X magnification from 10 randomly chosen fields within the same section of kidney tissue from an individual biopsy specimen [14 (link)]. The interstitial volume, α-smooth muscle actin (α-SMA) score were calculated by the point counting method in accordance with previous reports [16 (link), 18 (link)].
5
Western Blot Analysis of CYP1A1 and FOXP3
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The cells were washed twice with PBS and then lysed in RIPA Lysis Buffer (Beyotime, China) supplemented with a protease inhibitor cocktail (Medchem Express, Nanjing, China). The protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime, China). Quantitative protein samples (30 μg/lane) were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred to PVDF membranes. After blocking with 5% nonfat milk, the membranes were incubated overnight with anti-CYP1A1 antibody (Proteintech Group, Inc., 1:1000, 13241-1-AP), anti-FOXP3 antibody (Abcam, 1:1000, ab54501), and anti-GAPDH antibody (Bioworld Technology, Inc., 1:10000, AP0063) at 4˚C. Following incubation with horseradish peroxidase-conjugated goat anti‑rabbit antibodies (Bioworld Technology, Inc., 1:10000, BS13278), protein blotting was visualized using an Enhanced Chemiluminescence (Life Technologies, Carlsbad, USA). Data ware analyzed using Image-Lab 6.0.1 software (Bio-Rad).
6
Inducible FOXP3 Fusion Protein Analysis
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Example 95
A fusion molecule is generated by fusing full length or truncated (FOXP3A2) and DDs such as ecDHFR (DD) or FKBP (DD). These fusion molecules were cloned into pLVX-IRES-Puro vectors.
To test ligand dependent FOXP3 production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10% FBS and incubated overnight at 37° C., 5% CO2. Cells were transfected with the constructs using Lipofectamine 2000 and incubated for 24 hrs. Following the incubation, media was exchanged for growth medium with or without 10 μM Trimethoprim or 1 μM Shield-1 and further incubated for 24 hrs. Cells were harvested and FOXP3 levels were analyzed via western blotting using anti FOXP3 antibody (Abcam, Cambridge, UK). OT-FOXP3-003, and OT-FOXP3-007 showed the strongest Shield-1 dependent stabilization of FOXP3, while OT-FOXP3-008 showed the strongest Trimethoprim dependent stabilization of FOXP3 (
7
Quantitative Immunohistochemistry of Tumor-Infiltrating Lymphocytes
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A total of 20 tumor tissues and 10 normal tissues were obtained from patients in Nanfang Hospital. Subcutaneous tumor tissues of mice were collected from each group on day 30 after inoculation of tumor cells. Tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin sections (5 μm) were deparaffinized and rehydrated and treated with hydrogen peroxide and then carried out with antigen retrieval. The sections of patients were blocked for 10 minutes and then were incubated with anti‐FoxP3 antibody (1:3000; Abcam) for 1 hour, the sections of mouse tissues were incubated with anti‐mCD4 (1:700; Abcam), and anti‐mCD8 (1:1000; eBioscience) antibody according to the operation manual of rabbit‐specific HRP/DAB (ABC) Detection IHC Kit (Abcam). All tissues were counterstained with H&E.
8
Dual Immunohistochemical Staining Protocol
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Double immunostaining was performed according to the following methods. Sections of formalin-fixed, paraffin-embedded tissues (4 μm thick) were de-paraffinized, subjected to heat-mediated antigen retrieval in citric acid buffer at 98 °C for 40 min, and then blocked in 5% skim milk at room temperature for 1 h. They were incubated with anti-FOXP3 antibody (Abcam) overnight at 4 °C, followed by incubation with the appropriate secondary antibody (DAKO) at room temperature for 1 h, and then incubated with 3,3′-diaminobenzidine (Sigma-Aldrich) at room temperature for 5 min. After that, they were heated again in EDTA buffer (pH 9.0) in the same way. They were then blocked in 2.5% normal horse serum (ImmPRESS Horse Anti-Rabbit IgG Polymer kit; Vector Laboratories, Riverside, CA, USA) at room temperature for 20 min, followed by incubation with anti-CD3 antibody (Abcam) overnight at 4 °C. They were incubated with the secondary antibody (ImmPRESS Horse Anti-Rabbit IgG Polymer kit; Vector Laboratories) at room temperature for 30 min and then incubated with working solution prepared with Vector SG Peroxidase (HRP) Substrate Kit (Vector Laboratories) at room temperature for 5 min.
9
Histological and Immunohistochemical Analysis of Cardiac Tissue
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The excised hearts were fixed with 4% formalin for histological and immunohistochemical examinations. Following 24–48 h of dehydration, clearing, and embedding in paraffin wax, sliced Sects. (4 mm) were stained with haematoxylin and eosin and Masson’s trichrome and cardiac sections were dewaxed, subjected to antigen retrieval, and incubated with anti-IL17A antibody and anti-FOXp3 antibody (Abcam, Cambridge, UK), followed by incubation with a secondary antibody, and then analyzed using Image J (NIH V1.8.0.112).
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