IF staining was performed as described previously (51 (link), 52 (link)). After blocking, the slides were incubated with the primary antibodies to Ln-γ2 (#ab210959, Abcam; dilution ratio, 1:500), pan-cytokeratin (#ab7753, Abcam; dilution ratio, 1:400), α-SMA (#19245, Cell Signaling Technology; dilution ratio, 1:200), CD3 (#ab1669, Abcam; dilution ratio, 1:150 dilution), CD4 (#ab183685, Abcam; dilution ratio, 1:200 dilution), CD8 (#ab217344, Abcam; dilution ratio, 1:200 dilution), EpCAM (#36746, Cell Signaling Technology; dilution ratio, 1:100), p-c-Jun (Ser73) (#3270, Cell Signaling Technology; dilution ratio, 1:800), and p-c-Fos (Ser32) (#5348, Cell Signaling Technology; dilution ratio, 1:200) at 4°C overnight in a moist chamber. After thorough washing, the slides were then incubated with Alexa Fluor 594– or 488–conjugated secondary antibodies (Invitrogen). Last, all slides were mounted with antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; #S36938, Thermo Fisher Scientific). Images were captured with an OLYMPUS FV2000 fluorescence microscope.
Ab1669
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab1669 is a laboratory reagent produced by Abcam. It serves as a tool for scientific research and analysis. The core function of this product is to facilitate specific experiments and procedures within a laboratory setting. No further details on the intended use or interpretation of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab210959, by Abcam (1 mentions)
Ab7753, by Abcam (1 mentions)
Ab217344, by Abcam (1 mentions)
P c fos ser32, by Cell Signaling Technology (1 mentions)
Epcam, by Cell Signaling Technology (1 mentions)
Ab183685, by Abcam (1 mentions)
P c jun ser73, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 or 488 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
Anti cd3, by Abcam (1 mentions)
Cell lysis solution, by Beyotime (1 mentions)
Ab138515, by Abcam (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab150080, by Abcam (1 mentions)
Triton x 100, by Abcam (1 mentions)
Anti fade mounting solution, by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Microtome, by Leica (1 mentions)
6 protocols using ab1669
1
Immunofluorescence Staining of Tissue Sections
2
Immunohistochemical Analysis of Tissue Samples
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For the IHC analysis, the paraffin-embedded tissue was sectioned at a thickness of 3 μm and placed on poly-L-lysine-coated slides. The slides were dried overnight at 60°C. The sections were deparaffinized via two changes of xylene. After blocking endogenous peroxidase activity with 3% hydrogen peroxide in methanol, antigen retrieval was performed by heating the slides in 10 mmol/l citrate buffer (pH 6) using a water bath. The sections were then incubated with the primary antibody (anti-CD3, 1 : 200 dilution (Abcam, ab1669, Cambridge, MA, USA) and anti-FABP6, 1 : 500 dilution (Proteintech, 13781-1-AP, Wuhan, China)) at 37°C for 30 min. Thereafter, the sections were rinsed with Tris-buffered saline (TBS) three times, incubated with the secondary antibody at room temperature for 30 min, and washed with TBS. Finally, 3,3′-diaminobenzidine (DAB) was used to illuminate the positive staining signals, and hematoxylin was used for counterstain. The positive staining signals were classified into 4 grades according to standard procedures.
3
Western Blot Analysis of E2F2 and CCR4
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RASF transfected with anti-E2F2 siRNA or E2F2-expressing plasmids were homogenized with cell lysis solution (Beyotime, China) and centrifuged at 12,000 × γ for 30 min at 4oC. The supernatant was collected, and protein concentrations were determined using a BCA protein assay kit (Beyotime, China). Total protein was separated on 10% SDS-PAGE gels, transferred to PVDF membranes and blocked in 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at pH 7.5. The membranes were incubated overnight at 4oC with rabbit polyclonal antibody against human E2F2 (ab138515, Abcam, UK), goat polyclonal antibody against human CCR4 (ab1669, Abcam, UK) or rabbit polyclonal antibody against human GAPDH (Good Here, China) at a 1 : 1000 dilution. Horseradish peroxidase-conjugated sheep anti-rabbit immunoglobulin G (IgG) secondary antibody or rabbit anti-goat IgG secondary antibody was incubated with the membrane for 1 h at room temperature in TBST. Complexes were visualized using Immobilon Western chemiluminescent HRP substrate (Millipore, USA). The expression level of E2F2 or CCR4 protein was quantified by normalizing GAPDH expression using Image J software (NIH, Bethesda, MD, USA) to compare their gray values.
4
Immunohistochemical Staining of CD3 and CD68
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The tissue sections were deparaffinized and rehydrated in a series of ethanol (50–100% v/v). The heat‐induced epitope retrieval method was employed for 30 min using a citrate‐buffered antigen retrieval buffer (1×). The tissues were permeabilized with TBST (tris‐buffered saline + 0.25% Triton‐X‐100, Abcam, UK) and blocked with 10% w/v bovine serum albumin (Sigma–Aldrich MO, USA) for 2 h at RT. The slides were then incubated with primary antibodies, including anti‐CD3 rabbit monoclonal antibody (Abcam, ab1669) at a dilution of 1:200 in TBST and anti‐CD68 Alexa fluor‐488 labeled mouse anti‐rat monoclonal antibody (PIMA528262) at a dilution of 1:500 in TBST, for 4 h at RT. A secondary antibody to anti‐rabbit Alexa 594 (Abcam, ab150080) was used at a dilution of 1:500 in TBST. Nuclei were visualized by mounting sections with an antifade mounting solution containing DAPI (Vector Laboratories, USA). Images were captured using the Echo Revolution microscope.
5
Immunofluorescent Staining of Chemokines
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AT samples were fixed and mounted in paraffin and sectioned into 5 μm slides with a microtome (Leica Biosystems, Nussloch, Germany). Antigen was unmasked with proteinase K (cat#S3020, Dako, Santa Clara, CA) and blocked with 15% horse serum for 1 h. Samples were incubated with the following primary antibodies overnight at 4°C: mouse anti-human CCL17 (1:50, cat#DY364-05, R&D Systems), mouse anti-human CCL22 (1:50, cat#DY336, R&D Systems), goat anti-human CCR4 (1:100, ab1669; Abcam, Cambridge, UK), rabbit anti-human CD3 (1:100, cat#C7930, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-human CD31 (1:50, cat#ab32457, Abcam) and rat anti-human Mac-3 (1:100, cat#sc19991, Santa Cruz Biotechnology, Dallas, TX). Alexa-Fluor® 488 goat anti-rabbit (cat#A11034), Alexa-Fluor® 488 donkey anti-rat (cat#A21208), Alexa-Fluor® 594 goat anti-rat (cat#A1107), Alexa-Fluor® 594 goat anti-mouse (cat#A1105) and Alexa-Fluor® 594 chicken anti-goat (cat#A21468) antibodies were used as secondary antibodies (dilution 1:1000; all from Molecular Probes, Eugene, OR). Nuclei were stained with Hoechst (1:4000). Afterwards, five fields from each section were captured with a Zeiss Axio Observer A1 fluorescence microscope (Carl Zeiss Micro Imaging GmbH, Oberkochen, Germany).
6
Detecting Human T Cells in Murine Tumors
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Tumor tissues and organs were removed from mice, fixed in formalin, embedded in paraffin, and dissected into 3 mm-thick sections. Organ slides were stained with H&E. Fixed tumor slides were incubated with anti-human CD3 antibody (Abcam, Ab1669, 1:100) overnight at 4°C to detect the presence of human T cells in tumors. The slices were then treated with a secondary antibody and visualized using 3,3′-diaminobenzidine. The cell nuclei were stained with hematoxylin.
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