Paraffin-embedded specimens were cut into 5-μm sections, followed by IHC staining with anti-TBL1XR1 (1:400, HPA019182, Sigma), anti-Cyclin-D1 (1:200, 60186-1-Ig, Proteintech), anti-c-Myc (1:200, 10828-1-AP, Proteintech) and anti-E-cadherin (1:400, 20874-1-AP, Proteintech) antibodies using the method described before25 (link). IHC results were evaluated by two independent pathologists, according to both the staining intensity and percentage of positively stained cells. The staining intensity was defined as follows: 1 (negative staining), 2 (weak staining, slight yellow), 3 (moderate staining, dark yellow), 4 (strong staining, dark brown). The percentage of stained cells was scored as 1 (0–20% positive), 2 (21–50% positive), 3 (51–75% positive), 4 (76–100% positive). The final immunoreactivity score (IRS) was calculated by multiplying the average intensity score and percentage score (range 1–16), and protein expressions were identified as low-expression (IRS ≤ 6) and high-expression (IRS > 6) accordingly.
Hpa019182
Manufactured by Merck Group
HPA019182 is a laboratory equipment product offered by Merck Group. It serves as a core functional device for research and analytical applications. The detailed specification and intended use of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin 10366 1 ap, by Proteintech (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
β actin sc 1616, by Santa Cruz Biotechnology (1 mentions)
Twist1, by Proteintech (1 mentions)
C myc, by Proteintech (1 mentions)
Cyclin d1, by Proteintech (1 mentions)
2 protocols using hpa019182
1
Immunohistochemical Evaluation of Protein Expression
2
Evaluating Protein Levels via Western Blot
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Western Blot was performed to evaluate the protein levels as describe before28 (link). Briefly, cells were homogenized with NP-40 lysis buffer and then centrifuged the lysate at 12,000 rpm for 20 min to obtain supernatant. After protein concentration was determined by BCA protein assay kit (Thermo), equal amounts of total protein were resolved by 12% SDS-PAGE followed by transferring to PVDF membranes. Blots were blocked for 1 h in 5% BSA and then incubated overnight with the primary antibodies: TBL1XR1 (HPA019182, Sigma), Vimentin (10366-1-AP, Proteintech), Snai1 (13099-1-AP, Proteintech), Slug (12129-1-AP, Proteintech), TWIST1 (18125-1-AP, Proteintech), N-cadherin (13769-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), Cyclin D1 (60186-1-Ig, Proteintech), c-Myc (10828-1-AP, Proteintech) and β-actin (sc-1616, Santa Cruz). After washing three times with TBST, the membranes were incubated for another 1 h with secondary antibodies conjugated with horseradish peroxidase. The immunoreactivity was visualized using the enhanced chemiluminescence (ECL) reagent and autoradiographic film.
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