TMA slides were stained by IHC for ITGA3 integrin expression. 4-µm FFPE tissue sections were cut for each TMA. Slides were deparaffinized and pretreated with Heat-Induced-Epitope-Retrieval solution (pH 9) (DAKO). Slides are preincubated in Dual Endogenous Enzyme Block (DEEB) blocking solution in a DAKO Autostainer Link48. Primary antibody for ITGA3 (ab131055, dilution 1:200; Abcam) were used for IHC. The slides were incubated for 30 minutes and then washed, which was followed by detection by EDL solution (DAKO EnVision and Dual Link System-HRP). Hematoxylin was used as the counterstain. IHC slides were scored by two readers (QL and WM) and verified by two pathologists (TW and CZ) blinded to clinicopathologic information. Staining for ITGA3 was semi-quantified and assessed using both intensity and percentage of positive cells. Staining intensity was graded as 0= negative (no cells stained); 1= weak; 2= moderate; 3= strong. The percentage of positive cells was defined as 0 (0–5%), 1+ (6–20%), 2+ (21–50%), and 3+ (51–100%) (Figure 2A ). After the initial analysis, study pathologists recommended to measure the ITGA3 expression by tumor proportion score (TPS), i.e., the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. The specimen was considered to have ITGA3 expression if TPS >5% and no ITGA3 expression if TPS ≤5%.
Ab131055
Manufactured by Abcam
Sourced in United States
Ab131055 is a primary antibody product offered by Abcam. It is a polyclonal antibody targeting a specific protein. The core function of this product is to bind to and detect the target protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Autostainer link 48, by Agilent Technologies (1 mentions)
Ab133557, by Abcam (1 mentions)
Sc 7313, by Santa Cruz Biotechnology (1 mentions)
Cw0102, by CWBIO (1 mentions)
Cw0103, by CWBIO (1 mentions)
P5282, by Merck Group (1 mentions)
Flna antibody, by Cell Signaling Technology (1 mentions)
Ab182858, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab11220, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Dab kit, by ZSGB-BIO (1 mentions)
Ab6994, by Abcam (1 mentions)
Ab182120, by Abcam (1 mentions)
Ab182981, by Abcam (1 mentions)
7 protocols using ab131055
1
Immunohistochemical Analysis of ITGA3 Integrin
2
High-throughput Protein Detection Assay
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Total protein was isolated from cells by using lysis buffer (P0013B; Beyotime, China). For high-throughput protein detection, we cut the membrane corresponding to the protein molecular weight prior to incubation with antibodies. Immunoblotting was performed with primary antibodies against MUC1 (sc-7313; Santa Cruz, USA; 1:200), ITGA2 (ab133557; Abcam, USA; 1:500), ITGA3 (ab131055; Abcam, USA; 1:500), ERK1/2 (4695; CST, USA; 1:1000), phosphorylated ERK1/2 (Thr202/Tyr204) (4370; CST, USA; 1:500) and GAPDH (60004-1-Ig; Proteintech, China; 1:5000) as the control and followed by the secondary antibodies (CW0102S and CW0103S; CWbiotech, China; 1:5000). The specific bands were visualized with super enhanced chemiluminescence (ECL) detection reagent (180–5001; Tanon, China).
3
Quantitative Immunofluorescence Analysis of Integrin Proteins
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HCT116 cells were fixed with 4% formaldehyde for 15 minutes at room temperature. After washing the cells 3 times in PBS with 0.1% Triton X-100, cells were blocked with 4% bovine serum albumin for 30 minutes. Cells were incubated with primary ITGB4 antibody (ab182858, Abcam), ITGA3 (ab131055, Abcam), FLNA antibody (4762; Cell Signaling Technology, Danvers, MA, USA), FITC-phalloidin antibody (P5282, Sigma) and PLEC antibody (ab11220, Abcam) for 1 hour at room temperature. Following washes with PBS, 0.1% Triton X-100, cells were incubated with FITC/TRITC-conjugated secondary antibodies for 1 hour at room temperature. Following washes with PBS, 0.1% Triton X-100, cells were stained with DAPI (Sigma) and visualized by confocal scanning microscopy (710; Zeiss, Oberkochen, Germany). The original TIF images were quantified by immunofluorescence using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Signal and background intensity was measured. The net fluorescence intensity is obtained by subtracting the background intensity from the signal intensity.
4
Western Blot Analysis of Integrin α3
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Radio immunoprecipitation assay lysis buffer (Beyotime, China) was used to extract cell lysates. Quantified by the BCA protein assay kit (Beyotime, China), the proteins were separated by 12% SDS-PAGE and transferred onto the PVDF membrane. 5% skimmed milk powder was harnessed to keep the membrane sealed for 2 h; then, it was incubated overnight with primary antibodies at 4°C. Next, phosphate-buffered saline with Tween® was harnessed to rinse the membrane and it was cultured at room temperature with the secondary antibody for 2 h. Eventually, an enhanced chemiluminescence (ECL) kit (GE Healthcare, USA) was in use to detect protein signals. The primary antibodies rabbit anti-ITGA3 (ab131055, 0.3 μg/ml) and rabbit anti-GAPDH (ab181602, 1 : 10,000) and secondary antibody goat anti-rabbit IgG (ab205718) were provided by Abcam (China).
5
Quantifying Integrin Expression in Tissues
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The expression of integrin alpha 3 (ITGA-3) and integrin alpha 5 (ITGA-5) were compared stained with DAB staining and hematoxylin. Briefly, paraffin sections were dewaxed, and antigen was retrieved by citric acid buffer. The primary antibody of ITGA-3 and ITGA-5 is the rabbit antibody (Abcam, ab131055 and ab239400, respectively, 1 : 100). The corresponding secondary antibody is HRP-goat anti-rabbit IgG (H+L) (GXYbio, S8002, 1 : 100). The sections were reacted with the DAB kit (ZSGB-BIO, China, ZLI-9019). Eight separate views (magnification = original × 400) were randomly selected. The integrated option density (IOD) of ITGA-3 and ITGA-5 was chosen to determine the protein semiquantitative expression. Free ImageJ software (version 1.2; WS Rasband, National Institute of Health, Bethesda, MD) was used to conduct deconvolution and downstream analysis [22 (link)], and then, the Mann-Whitney test of the IOD was performed using SPSS software (version 25.0, IBM).
6
Immunofluorescence Staining of Endothelial Markers
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For immunofluorescence, cells were fixed with 4% formaldehyde at room temperature for 25 minutes, followed by three 10-minute washes in PBS. Permeabilization was achieved with 0.2% Triton X-100 (#85111, Thermo Fisher Scientific, USA) for 3 minutes, and non-specific binding was blocked using 5% BSA for 20 minutes. The primary antibody was applied overnight at 4°C. Afterward, cells were incubated with the secondary antibody for 2 hours at room temperature, then washed three times with PBS (#10010023, Gibco, USA). The cells were subsequently examined under a fluorescence microscope to visualize staining. The following antibodies were used: Anti-CD31 antibody (#ab182981, 1:800, Abcam, UK), anti-Von Willebrand Factor antibody (#ab6994, 1:800, Abcam, UK), anti-integrin beta 4 antibody (#ab182120, 1:1000, Abcam, UK), anti-Integrin alpha 3 antibody (#ab131055, 1:1000, Abcam, UK), anti-LAMB3 antibody (ab97765, 1:1000, Abcam, UK), anti-Laminin alpha 5 antibody (#ab210957, 1:1000, Abcam, UK). The staining intensity was evaluated with ImageJ Software.
7
Immunohistochemical Analysis of ITGA3
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Sections of 3-μm thickness were used for immunostaining. A rabbit polyclonal antibody against human ITGA3 (ab131055; Abcam, Cambridge, MA, USA) was used for immunohistochemistry (IHC) as described previously [19 (link)]. The IHC score was calculated by judging the percentage of positively stained cells. The scores ranged from 0 to 3 (0, 0%; 1, 5%; 2, 5–50%; and 3, >50% immunoreactive cells). Scores of 0 or 1 were considered to indicate “low” expression, while scores of 2 or 3 indicated “high” expression of ITGA3.
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