Murine colon tissue sections were blocked with 3% H2O2, 2% BSA in 0.01 M PBS (pH 7.3) for 30 min. After overnight incubation at 4°C with the anti-IL-6 antibody (1:300; rabbit, Abcam) or anti-CD4 antibody (1:1000; rabbit, Abcam), immunofluorescence of CD4 and IL-6 was performed using a Three-Color Fluorescence kit (Shanghai Recordbio Biological Technology, Shanghai, China) for 10–15 min at room temperature, based on the tyramide signal amplification (TSA) technology. After extensive washing, the nuclei were stained with DAPI. Negative controls were run concurrently, except that the antibody dilution buffer was used to substitute the primary antibody (21 (link)). They were scanned with a Leica SP5 (Mannheim, Germany) confocal laser scanning microscope equipped with a ×63 objective, and then with three times magnification. Three channels were used to acquire the images sequentially: FITC laser (488 nm, green for IL-6), Cy5 laser (633 nm, rose for CD4), and DAPI (405 nm, blue for nuclei). We captured images separately and then merged them. The cells showing co-localization of signals generated from both the green (IL-6) and rose (CD4) channels were identified as IL-6 + CD4 + T cells.
Anti cd4 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-CD4 antibody is a laboratory reagent used for the detection and analysis of CD4 protein expression. CD4 is a glycoprotein found on the surface of T helper cells and plays a crucial role in the immune response. This antibody can be used in various immunoassays and cell-based applications to study the expression and distribution of CD4 in biological samples.
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Lab products found in correlation
Anti cd8 antibody, by Abcam (4 mentions)
Anti cd3 antibody, by Abcam (3 mentions)
Anti cd8 antibody, by Novus Biologicals (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti il 6 antibody, by Abcam (1 mentions)
Lsm 510 laser confocal microscope, by Zeiss (1 mentions)
Tissuefaxs platform, by TissueGnostics (1 mentions)
Anti cd68 antibody, by Abcam (1 mentions)
Leica dm5500b microscope, by Leica Microsystems (1 mentions)
Neutral buffered formalin, by Thermo Fisher Scientific (1 mentions)
Anti mouse foxp3 antibody, by Abcam (1 mentions)
Anti flag antibody, by ABclonal (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Anti gfp antibody, by ABclonal (1 mentions)
Anti β actin antibody, by Beyotime (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Anti il21r 4a9, by BioXCell (1 mentions)
Anti mouse total erk1 2, by Cell Signaling Technology (1 mentions)
13 protocols using anti cd4 antibody
1
Multicolor Immunofluorescence of IL-6 and CD4
2
Immunofluorescence Analysis of Thyroid Gland
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Some thyroid gland sections were stained with haematoxylin and eosin (H & E),
while the remaining sections were incubated with the primary
anti-thyroid-hormone-receptor β1 (TRβ1) antibody (1:200) or anti-vitamin D
receptor (VDR) antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA,
USA), and anti-CD4 antibody (1:200; Abcam, Cambridge, MA, USA), at 4°C
overnight. The following day, the sections were incubated with secondary
antibodies in the dark for 30 min at 37°C. The nuclei were stained with 100 μl
Hoechst 33258, and the sections were visualized using a Zeiss LSM 510 laser
confocal microscope (Carl Zeiss Microscopy GmbH, Germany) for immunofluorescence
analysis.
while the remaining sections were incubated with the primary
anti-thyroid-hormone-receptor β1 (TRβ1) antibody (1:200) or anti-vitamin D
receptor (VDR) antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA,
USA), and anti-CD4 antibody (1:200; Abcam, Cambridge, MA, USA), at 4°C
overnight. The following day, the sections were incubated with secondary
antibodies in the dark for 30 min at 37°C. The nuclei were stained with 100 μl
Hoechst 33258, and the sections were visualized using a Zeiss LSM 510 laser
confocal microscope (Carl Zeiss Microscopy GmbH, Germany) for immunofluorescence
analysis.
3
Multicolor Immunohistochemistry Analysis
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The 5 um non-stained formalin-fixed and paraffin-embedded slides were applied for immune staining. Paraffin sections were then placed in a 70°C paraffin oven for 1 hour, followed by deparaffinized in xylene and then rehydration. Ag was recovered with citric acid buffer (pH 6.0) in the oven for 60 minutes. Inactivate endogenous peroxidase by incubation in 3% H2O2 for 15 minutes. After preincubation with 10% normal goat serum to block nonspecific sites for 10 minutes, sections are incubated overnight with primary antibodies including anti-CD8 antibody (Abcam, cat.189926), anti-CD4 antibody (Abcam, cat.133616), anti-Anti-Osteopontin antibody (Abcam, cat.269411), anti-CD68 antibody (Abcam,cat.213363), anti-BCA1 (Abcam, cat.246518), and antibody CD19 (Abcam, cat.134114) in a humidified chamber at 4°C. Secondary HRP-conjugated antibodies were added and incubated at room temperature for 10 minutes. For multicolored IHC analysis, fluorophore-conjugated TSA were used after incubation with secondary antibodies. After washing with PBS twice, the images were visualized using the TissueFAXS platform (TissueGnostics).
4
Immunohistochemical Analysis of hAAT Expression
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Immunohistochemical staining to detect hAAT within myofibers as well as H&E staining was performed after tissues were fixed in 10% neutral buffered formalin for 24 h at room temperature (Fisher Scientific) and embedded in paraffin by University of Massachusetts Chan Medical School Morphology Core (Worcester, MA). Anti-AAT antibody (Fitzgerald, Acton, MA), anti-CD4 antibody (Abcam), and anti-mouse FOXP3 antibody (Abcam) were used as primary antibody. Images were acquired on a Leica DM5500 B microscope (Leica Microsystems, Wetzlar, Germany).
5
Immunoblot Analysis of Protein Expression
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3D4/21 cells were plated in six-well culture dishes and transfected with target vectors when they converged to approximately 70%. After 24 h and 48 h, the total proteins in the cells were extracted using RIPA buffer (Thermo Fisher Scientific, New York, USA, #89900). The proteins were diluted in SDS loading buffer and then heated for 10 min at 100 °C for denaturation. The proteins were electrophoresed through 12% SDS-PAGE and then transferred to a nitrocellulose filter. Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated with 1:1000 anti-GFP antibody (ABclonal, AE012), 1:2000 anti-flag antibody (ABclonal, AE005), 1:1000 anti-CD4 antibody (Abcam, UK, 25804), 1:1000 anti-β-actin antibody (Beyotime, Shanghai, China, AF0003), or 1:1000 anti-p-p65 antibody (CST, USA, 4025) at 4 °C overnight. Membranes were incubated with 1:1000 horseradish peroxidase-labeled anti-rabbit-IgG secondary antibody (Beyotime, Shanghai, China, A0208) or anti-mouse-IgG secondary antibody (Beyotime, Shanghai, China, A0216) for 1 h at room temperature. Immunodetection was performed by an Image Quant LAS4000 mini (GE, USA). The gray values of the protein bands were measured using ImageQuantTL software (GE, USA), and β-actin protein levels were used as internal control.
6
Multiparameter Flow Cytometry Analysis
7
Immunohistochemical Analysis of Tumor Microenvironment
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Immediately after mice were sacrificed, tumor tissue was fixed in 10% formalin before paraffin embedding. Standard procedures were used for hematoxylin and eosin (H&E) staining. For immunostaining, formalin-fixed, paraffin-embedded tissue was treated with xylene, rehydrated with ethanol, and heated in a microwave with citric buffer to retrieve antigens. For blocking purpose, the tissues were incubated for 30 minutes, with 5% bovine serum albumin buffer. Followed by overnight incubation at 4° C, with primary antibodies: anti-CD3 antibody, anti-CD4 antibody, and anti-NKp46 antibody (Abcam, Cambridge, United Kingdom) at 1:100 dilutions, anti-CD8 antibody (Novus Biologicals, Minneapolis, MN) at 1:20 dilution, anti-CD31 (Novus Biologicals, Minneapolis, MN) at 1:100, anti-F4/80 (R&D system Minneapolis, MN) at 1:100, anti-CD206 (R&D system Minneapolis, MN) at 1:100, as well as anti-CD80 (Novus Biologicals, Minneapolis, MN) at 1:100. After washing, tissues were incubated with fluorescence-conjugated secondary antibodies at room temperature for 1 hr. Slides were prepared with antifade mountant with 4’,6-diamidino-2-phenylindole (DAPI).
8
Quantifying T-cell Subtypes in Spinal Cord Tissue
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The cross-sections (5 mm thick) were dewaxed using xylene and dehydrated in a graded series of alcohols after incubation at 60°C for 1 h. The endogenous peroxidase activity was quenched with 3% H2O2, and heat-induced epitope retrieval was done in sodium citrate buffer. Sections were incubated with anti-CD3 antibody (Abcam, Cambridge, UK), anti-CD4 antibody (Abcam, Cambridge, UK), and anti-CD8 antibody (Abcam Cambridge, UK) overnight at 4°C, followed by incubation with SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (Cell Signaling Technology, Danvers, MA, USA) according to instructions from manufacturers. Final color product was developed with SignalStain DAB Substrate Kit (Cell Signaling Technology, Danvers, MA, USA), and then sections were counterstained with hematoxylin (Leagene, Beijing, China). Images were captured by LEICA DM6000B with a LEICA DFC300 FX (Leica Microsystems Ltd., Solms, Germany) at a magnification of 200x. Six fields were evaluated for each slide [25 (link)]. The numbers of positive cells per mm2 of spinal cord tissues were made by manual counting at Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) [26 (link)].
9
Liver Histology and Immunohistochemistry Protocol
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Liver histology (H&E staining) and the NAFLD activity score were assessed as previously described.[26] (link), [27] , [28] Sirius Red or Trichrome staining, provided by a Pathology Core at the University of Kentucky, was used to analyze liver fibrosis. For liver immunohistochemical staining, liver paraffin sections were stained with anti-CD41 antibody (Thermo Fisher Scientific; MA, USA), anti-neutrophil antibody [7/4] (ab53457) (Abcam; Cambridge CB20AX, UK), anti-p-selectin antibody (Novus Biologicals, CO, USA), anti-CD4 antibody (Abcam; Cambridge CB20AX, UK), or anti-CD8 antibody (Novus Biologicals, CO, USA), followed by incubation with biotinylated secondary antibody, peroxidase substrate diaminobenzidine (Vector Lab) and counterstaining with hematoxylin. Images were captured by a Nikon Eclipse 55i microscope. Semi-quantification of neutrophil, CD4, or CD8 positive cells in liver sections was performed as previously described.29 (link)
10
Immune Cell Profiling by Flow Cytometry
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The rate of CD4+CD25+ T cells, NK cells, and T cells subgroup were all measured by flow
cytometry. The following antibodies were used in our study: anti-CD4 antibody (Abcam,
Cambridge, UK), anti-CD25 antibody (Abcam, Cambridge, UK), anti-CD127 antibody (Abcam,
Cambridge, UK), anti-CD3 antibody (Abcam, Cambridge, UK), anti-CD16 antibody (Abcam,
Cambridge, UK), anti-CD56 antibody (Abcam, Cambridge, UK), anti-CD8 antibody (Abcam,
Cambridge, UK), and anti-CD45 antibody (Abcam, Cambridge, UK). Flow cytometry analysis was
performed on a FACScan (BD Biosciences, Mountain View, California) using CellQuest
software.
cytometry. The following antibodies were used in our study: anti-CD4 antibody (Abcam,
Cambridge, UK), anti-CD25 antibody (Abcam, Cambridge, UK), anti-CD127 antibody (Abcam,
Cambridge, UK), anti-CD3 antibody (Abcam, Cambridge, UK), anti-CD16 antibody (Abcam,
Cambridge, UK), anti-CD56 antibody (Abcam, Cambridge, UK), anti-CD8 antibody (Abcam,
Cambridge, UK), and anti-CD45 antibody (Abcam, Cambridge, UK). Flow cytometry analysis was
performed on a FACScan (BD Biosciences, Mountain View, California) using CellQuest
software.
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