IHC was performed by using human breast cancer microarrays of formalin-fixed paraffin-embedded (FFPE) tissues (Alianna, Xi an, China), and isolated fibroblasts were stained with antibodies against human α-smooth muscle actin (α-SMA) (ab5694; Abcam, Cambridge, UK) and FAP (ab28244; Abcam). Antibodies (1:100 dilutions) were incubated at 4 °C overnight. Antibody staining was developed using the Vectastain ABC kit (#PK-4000) and DAB (#SK-4100) detection system (Vector Laboratories, CA) and accompanied by hematoxylin counterstaining. Scoring for each immunohistochemistry marker was performed by two experienced technologists who were blinded to the results of other markers or case identity.
Ab28244
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab28244 is an antibody used for research purposes. It is a polyclonal antibody targeted against a specific protein. The core function of this product is to detect and bind to the target protein in research samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab5694, by Abcam (7 mentions)
Ab32575, by Abcam (6 mentions)
Ab124964, by Abcam (5 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Ab16667, by Abcam (2 mentions)
Ab92547, by Abcam (2 mentions)
Ab28364, by Abcam (2 mentions)
Ab7817, by Abcam (2 mentions)
Ab2413, by Abcam (2 mentions)
Ab71916, by Abcam (2 mentions)
Sab3500533, by Merck Group (2 mentions)
Ts100 f, by Nikon (2 mentions)
Ab6717, by Abcam (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Ab134045, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Keygen Biotech (1 mentions)
31 protocols using ab28244
1
Immunohistochemical Analysis of Cancer-Associated Fibroblasts
2
Protein Expression Analysis of Exosomes
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Exosomes and cells were lysed using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (KeyGen Biotech) and centrifuged at 4°C for 5 min. Protein was separated on SDS-PAGE gel with various concentrations depending on the molecular weight of the protein and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). To detect the indicated proteins, primary antibodies were used as follows: vimentin (Cell Signaling Technology, 5741S, 1:1,000), FAP (Abcam, ab28244, 1:1,000), α-SMA (Abcam, ab5694, 1:500), CD63 (Abcam, ab134045, 1:1,000), CD81 (Abcam, ab79559, 1:1,000), and GAPDH (Cell Signaling Technology, #2118, 1:1,000).
3
Immunohistochemical Profiling of Tumor Microenvironment
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The cancer samples were labeled for PS1 (NB100-74510, Novus Biologicals, Centennial, CO, USA; PA5-13214, Invitrogen), FAP (ab28244, Abcam), α-SMA (ab32575, Abcam), fibroblast-specific protein (FSP) (ab41532, Abcam), and anti-CD8 (ab85792, Abcam) by immunohistochemistry. Primary antibodies were added to each section. The sections were covered with 4plus biotinylated goat antirabbit IgG (Biocare Medical, Pacheco, CA, USA) and incubated in a humidified chamber for 30 min at room temperature. Primary tumors excised from mouse xenografts were snap frozen for subsequent histological examination. Images were collected using a Leica DFC310 FX microscope (Buffalo Grove, IL, USA). The stained sections were evaluated for the number of CD8+ positive cells using anti-CD8a antibody (550,298, BD Biosciences, Franklin Lakes, NJ, USA). Scoring was divided into the following categories: <5 cells/high-power field (HPF; 40 × magnification), 5–10 cells/HPF, 10–15 cells/HPF, 15–30 cells/HPF, and >30 cells/HPF. The topographic distribution of positive cells was also evaluated. Location relative to the neoplastic follicle (perifollicular, intrafollicular, or neither, i.e., evenly distributed) was recorded.
4
Western Blot Analysis of Cell Signaling
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The cellular lysates were prepared according to the authors’ previous instructions. Briefly, total protein lysates were obtained via NP40 lysis buffer. The protein concentration was detected by BCA assay (Bio‐Rad). Proteins were resolved under 10% SDS‐PAGE conditions, then transferred to polyvinylidene difluoride blotting membranes (PVDF) membranes (Immobilon, Bedford, MA). The membranes were blocked with 5% BSA for 1 h at room temperature, then incubated with primary antibody Ki67 (ab16667, Abcam), HIF‐1α (ab179483, Abcam), EGFR (A11351, Abclonal), Anti‐Fibroblast activation protein (FAP, ab28244, Abcam), PDGFR (ab16667, Abcam), E‐cadherin (ab40772, Abcam), β‐catenin (ab16051, Abcam), and β‐actin (A5441, Sigma‐Aldrich, St. Louis, MO, USA) at 4 °C overnight. After three washes with TBST (20 mm Tris, 150 mm NaCl, 0.1% w/v Tween‐20), the membranes were subsequently incubated with the horseradish peroxidase‐conjugated secondary antibodies for 1 h at room temperature. Finally, three washes with TBST and visualized by enhanced chemiluminescence using ImageQuant LAS 4000 (GE Healthcare, Pewaukee, WI).
5
Immunohistochemical Analysis of Tissue Markers
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Deparaffinized tissues were heated to expose the antigens using Tris-ethylene diamine tetraacetic acid (EDTA) retrieval solution (pH 6.0) at 95°C for 30 minutes. Wash with PBS for three times per 3 minutes and incubate the sections in 3% hydrogen peroxide (H2O2) to block endogenous peroxidase activity. And then, samples were stained using primary antibodies to detect α-SMA (Abcam, ab32575), FAP (Abcam, ab28244), vimentin (Abcam, ab92547), fibronectin (Abcam, ab45688), E-cadherin (Abcam, ab76319), VCAM-1 (Abcam, ab134047), collagen I (Abcam, ab138492), cytokeratin (Abcam, ab6401). Antibodies were followed by means of peroxidase-conjugated secondary antibody and revealed with 3-39-diaminobenzidine (DAB) as chromogen.
6
Immunofluorescent Staining of Mesenchymal Markers
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Cells were seeded in 24-well plates and incubated overnight. After the indicated treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% bovine serum albumin. The cells were then incubated with anti-human antibody against vimentin (Cell Signaling Technology, 5741S, 1:100), FAP (Abcam, ab28244, 1:100), and α-SMA (Abcam, ab5694, 1:200) overnight at 4°C, followed by incubation with Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibody (Cell Signaling Technology) at room temperature for 30 min. Cells were observed and imaged using a fluorescence microscope (Nikon Eclipse Ti, Japan).
7
Immunohistochemical Evaluation of EGFR and Fibroblast Density in Tumor Samples
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Tumor samples were fixed in formaldehyde and paraffin-embedded. Immunohistochemistry for EGFR was performed on tumors slices and counterstained. Hematoxylin/Eosin (H&E) staining was performed on tumor slices. Fibroblast density was calculated as area per High-Power Field (HPF). For immunofluorescence on 3T3MEFs, cells were plated onto round, poly-L-lysine-coated glass coverslip in 24-well plates. YUMM conditioned media (CM) was generated by culturing cells to 100% confluency and then replacing media for FBS-free RPMI. CM was collected after 48 h, fractioned and at −80 °C. Cells were left unstimulated, or treated with 5 ng/mL TGF-β1 (Peprotech, Rocky Hill, CY, USA) or YUMM conditioned media, for 24 h. Then cells were fixed in methanol, stained with anti-SMA AF488 (ab184675, Abcam, Cambridge, UK) and anti-FAP (ab28244, Abcam, Cambridge, UK) overnight at 4 °C. Then they were incubated with Goat anti-Rabbit PE and Hoechst. Coverslips were mounted and analyzed in a fluorescent microscope.
8
Immunofluorescence Analysis of EMT and Fibroblast Markers
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For immunofluorescence analysis, the monocultures and co-culturing cells were seeded on 35 mm2 glass-bottom dishes covered with type-1 rat tail collagen in a growth medium. After 5 days of culturing, the expression of EMT markers and fibroblast-activated proteins was assessed by an immunocytochemical method using antibodies to E-cadherin (ab15148, Abcam, Cambridge, MA, USA), vimentin (ab16700, Abcam, Cambridge, MA, USA), FAP (ab28244, Abcam, Cambridge, MA, USA), αSMA (ab5694, Abcam, Cambridge, MA, USA), EpCam (ab20160, Abcam, Cambridge, MA, USA), and secondary antibodies conjugated with a fluorescent label, either Fluorescein isothiocyanate (FITC; ab6825, Abcam, Cambridge, MA, USA) or Alexa (ab6825, Abcam, Cambridge, MA, USA). Staining was performed according to the antibody manufacturer’s protocols, while the fluorescent dye DAPI was used for staining the nuclei. Fluorescence was recorded using a Leica DMIL fluorescence microscope (Leica, Wetzlar, Germany) equipped with filters: A4 UV BP 360/40 400 BP 470/40 for DAPI, CFP ET YFP ET (Ex: BP 500/20, Em: BP 535/30) for FITC and TX2 green BP 560/40 595 BP 645/75 for Alexa.
9
Integrin Beta-6 Monoclonal Antibody Protocol
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The mouse-anti-human monoclonal antibody R6G9 (IgG2a), which is directed against the extracellular domain of human integrin subunit β6, was obtained from Chemicon International (Temecula, CA, U.S.A.). The following monoclonal antibodies were obtained from Abcam (Cambridge, MA, U.S.A.): ab5694 and ab28244, which target the CAF markers α-SMA and FAP, respectively: ab27969, which targets transforming growth factor β (TGF-β); and ab9797, ab9695, ab16828, and ab6672, which target the four cytokines stromal cell-derived factor-1 (SDF-1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and interleukin 6 (IL-6). EP1186Y and EP1254 monoclonal antibodies, which target matrix metalloproteinase (MMP) 3 (MMP-3) and MMP-9, respectively, were also purchased from Abcam. Reagents for SDS/PAGE and molecular weight markers were obtained from Bio-Rad Laboratories (Hercules, CA, U.S.A.). The C–X–C chemokine receptor type 4 axis (CXCR4) antagonist AMD3100 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).
10
Uterine Tissue Histological Analysis
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For hematoxylin and eosin staining, the uterus was fixed overnight in 4% paraformaldehyde. Then, it was embedded in paraffin, sectioned, and stained.
For immunohistochemistry analysis, the paraffin blocks were sectioned and blocked with a blocking buffer. Immunostaining was performed using the primary antibodies estrogen receptor (ER; anti-rabbit, ab3206, Abcam), progesterone receptor (PR; anti-rabbit, ab16661, Abcam), and fibroblast activation protein (FAP; anti-rabbit, ab28244, Abcam) and visualized using goat anti-rabbit antibody (SP-9001, Beijing Zhong Shan Golden Bridge Biotechnology, China) and 3,3′-diaminobenzidine stain.
For immunohistochemistry analysis, the paraffin blocks were sectioned and blocked with a blocking buffer. Immunostaining was performed using the primary antibodies estrogen receptor (ER; anti-rabbit, ab3206, Abcam), progesterone receptor (PR; anti-rabbit, ab16661, Abcam), and fibroblast activation protein (FAP; anti-rabbit, ab28244, Abcam) and visualized using goat anti-rabbit antibody (SP-9001, Beijing Zhong Shan Golden Bridge Biotechnology, China) and 3,3′-diaminobenzidine stain.
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