All formalin-fixed and paraffin-embedded tissue samples were cut into 5-µm-thick sections. Immunohistochemical staining was performed using a standard immunoperoxidase staining procedure (anti-PLCε; dilution 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; anti-Gli-1, dilution 1:200 and anti-Gli-2, dilution 1:150; both were from Abcam, Cambridge, UK). The expression status of immunostaining was reviewed and scored based on the proportion of positive cells and staining intensity by a pathologist. Staining intensity was scored as follows: 0 (no staining); 1 (light yellow); 2 (light brown); 3 (brown); and 4 (deep brown). Immunoreactivity ratio was scored as follows: 0 (0% immunoreactive cells), 1 (<5% immunoreactive cells), 2 (5–50% immunoreactive cells), 3 (>50–75% immunoreactive cells) and 4 (>75% immunoreactive cells). The final immunoreactivity score was defined as the sum of both parameters. Final scores of ≤1 were regarded as negative expression, while scores of ≥2 were regarded as positive.
Anti gli2
Manufactured by Abcam
Sourced in United Kingdom
Anti-Gli2 is a laboratory equipment product that detects the Gli2 protein. Gli2 is a transcription factor involved in the Hedgehog signaling pathway. This product can be used in various research applications to study the Gli2 protein and its role in cellular processes.
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Lab products found in correlation
Anti gli1, by Abcam (2 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Anti pan akt, by Cell Signaling Technology (1 mentions)
Anti pakt s473, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti sonic hedgehog, by Abcam (1 mentions)
Anti pakt t308, by Cell Signaling Technology (1 mentions)
Anti vegfa, by Abcam (1 mentions)
Anti gli3, by Abcam (1 mentions)
Gapdh, by Proteintech (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
4 protocols using anti gli2
1
Immunohistochemical Analysis of Signaling Pathways
2
Western Blotting Analysis of Cell Signaling
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Western blotting was performed as described previously27 (link). Briefly, tissue or cell samples were lysed on ice in RIPA lysis buffer containing 1% PMSF (Beyotime, China). Total protein samples (40 μg) were separated by SDS-PAGE and then transferred onto a PVDF membrane. Primary antibodies were used as follows: anti-E-cadherin (Cell Signaling, 3195), anti-VEGF-A (Abcam, ab46154), anti-Sonic Hedgehog (Abcam, ab53281), anti-Gli2 (Abcam, ab2605556), anti-Gli3 (Abcam, ab6050), anti-p-AKT T308 (Cell Signaling, 4056), anti-p-AKT S473 (Cell Signaling, 4046), anti-pan-AKT (Cell Signaling, 4691), Beclin1 (Cell Signaling, 3495), and anti-LC3B (Sigma-Aldrich, L7543). GAPDH (Proteintech, 600004-1-Ig) or β-actin (Cell Signaling, 4970) was used as an internal standard. Immunofluorescent anti-rabbit and anti-mouse secondary antibodies were purchased from LI-COR Bioscience (Lincoln, USA), and the signals were visualized with an Odyssey Infrared Imaging System (Lincoln, USA). Anti-rabbit-HRP and anti-mouse-HRP secondary antibodies were purchased from Beyotime (Shanghai, China). After ECL exposure, images were captured by an Amersham Imager 600 (GE, USA). ImageJ software was used to quantify the immunoreactive bands.
3
Protein Expression Analysis in THCA Cells
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Protein was isolated from THCA cells using RIPA buffer (Beyotime, Nanjing, China). Proteins were separated using 10% SDS-PAGE and later transferred onto PVDF membrane (Millipore, Boston, USA). Membranes were blocked with 5% skim milk, then incubated with the primary antibodies anti-OCT4 (Cell Signaling Technology, Danvers, MA, USA), anti-NANOG (Cell Signaling Technology), anti-GLI2 (Abcam, Cambridge, UK), and anti-β-Actin (Proteintech, Wuhan, China) for 8 hours at 4 °C. Horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signaling Technology) was used as the secondary antibody. An enhanced chemiluminescence detection kit (Invitrogen) was used for blot detection.
4
Hedgehog Signaling Pathway Analysis
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Liver tissues were homogenized and digested in 1×RIPA buffer containing a protease inhibitor and phosphatase inhibitor cocktail solution (Thermo Scientific, Waltham, MA, USA). Western blotting experiments were performed following a standard protocol. The primary antibodies used were anti-SHH (SC-9024; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Gli1 (ab167388; Abcam, Cambridge, UK), anti-Gli2 (ab26056; Abcam, Cambridge, UK), and anti-GAPDH (# 2118; Cell Signaling Technology, Danvers, MA, USA).
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