The ameloblastoma paraffin sections were stained using a multi-labeled immunohistochemistry staining kit (Akoya, Cat#NEL811001KT) according to the manufacturer’s protocols. Briefly, the sections were incubated in 3% H2O2 for 10 minutes for the first time. Every time the sections were incubated with primary antibodies, they were first subjected to heat-induced epitope recovery and blocked with 5% BSA. The HRP conjugate and three wavelengths (520, 570, and 650 nm) were then utilized to attach the different primary antibodies, which included PANCK (Proteintech, Cat#26411-1-AP; 1:1 000), CD3 (Proteintech, Cat#17617-1-AP; 1:800), CD34 (Abcam, Cat#ab81289; 1:800), CD68 (Abcam, Cat#ab213363; 1:800), FAP (Cell Signaling Technology, Cat#E1V9V; 1:800), ODAM (Affinity, Cat#DF13204; 1:800), KI67 (Novus, Cat#NB500-170; 1:800), SFRP1 (Abcam, Cat#ab126613; 1:800), HLA-B (Thermo Fisher Scientific, Cat#PA5-35345; 1:800), LAMC2 (Abcam, Cat#ab210959; 1:800), EZH2 (Cell Signaling Technology, Cat#5246 S; 1:800), and KRT13 (Proteintech, Cat#10164-2-AP; 1:800). Finally, the cell nuclei were stained with DAPI (Solarbio, Cat#C0065-50ml).
Lamc2
Manufactured by Abcam
Sourced in United Kingdom
LAMC2 is a protein that is a component of the laminin protein complex. Laminins are a family of extracellular matrix glycoproteins that are a key structural component of the basement membrane. LAMC2 is a necessary subunit for the formation of the laminin-332 isoform.
Automatically generated - may contain errors
Lab products found in correlation
Sfrp1, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Eif4a3, by Proteintech (1 mentions)
Gapdh, by Abcam (1 mentions)
Col6a1, by Abcam (1 mentions)
Itgb4, by Abcam (1 mentions)
Anti angpt2, by Abcam (1 mentions)
Image lab software version 5, by Bio-Rad (1 mentions)
Itgb8, by Abcam (1 mentions)
Fgf18, by Abcam (1 mentions)
Lama3, by Abcam (1 mentions)
Itga2, by Abcam (1 mentions)
Ecl reagent, by CWBIO (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
4 protocols using lamc2
1
Multiplex Immunohistochemistry of Ameloblastoma
2
Protein Expression Analysis Workflow
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Cells were harvested and lysed by 1 × SDS buffer. Lysates were sonicated and centrifuged (13,000 rpm, 4 °C) for 10 min. Proteins were separated by 8–12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were immunoblotted with anti-rabbit NOP58 (1:1000), EIF4A3 (1:1000) (Proteintech, Chicago, USA; Abcam, UK) and anti-mouse LAMC2 (1:500), GAPDH (1:2000) (Abcam, UK; Zsbio, Beijing, China), and then were incubated with hybrid secondary antibody, and the data was collected by FluorChem V2.0 (Alpha Innotech Corp, USA).
3
Immunohistochemical Analysis of PCDH8 and LAMC2
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A GA tissue microarray was subjected to immunohistochemistry assays using a MaxVision HRP‐Polymer Detection System (Maixin, Fuzhou, China), as described previously.10 Primary antibody against PCDH8 (Santa Cruz Biotechnology, Paso Robles, CA, USA) and laminin subunit γ2 (LAMC2) (Abcam, Cambridge, MA, USA) were used according to the manufacturers’ guidelines. Slides were independently evaluated by 2 investigators who were blinded to the patients’ clinical information. The staining intensity of PCDH8 membrane immunostaining was evaluated (score 0 = none; 1 = weak; 2 = moderate; 3 = strong). Samples that scored ≥2 were regarded as high expression.
4
Western Blot Protein Analysis Protocol
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Total protein was extracted from cells using lysis buffer, followed by determination of total protein concentration in the sample. Each protein sample (50 µl) was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes. Membranes were blocked using 5% non-fat milk for 30 min at room temperature, then incubated with primary antibodies (1:1000 dilution; anti-ANGPT2, CD19, COL4A3, FGF18, ITGB4, ITGB8, LAMA3, LAMC2, PPP2R2C, SGK2, SYK, AKT3, COL6A1, CSF3, FGF1, ITGA2, ITGA11, MYB, PCK2, PGF, PIK3AP1, SGK1, TLR4 and TP53 (all Abcam, Cambridge, UK) at 4 °C overnight. Membranes were washed three times using 1 × Tris-buffered saline-Tween-20 (TBST), and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution; all Abcam) for 1 h at 37 °C, and again washed three times with 1 × TBST. GAPDH was quantified as the loading control. The blots were visualized using enhanced chemiluminescence (ECL) reagent (Cwbiotech, Beijing, China) and Image Lab software, version 5.1 (Bio-Rad Laboratories, Hercules, CA, USA).
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