The expression and cellular distribution of AFP and CXCR4 protein were assessed by immunohistochemical analysis. Five-millimeter-thick paraffin sections were deparaffinized and re-hydrated according to standard protocols, and heat-induced antigen retrieval was performed in sodium citrate buffer (10mmol/L, pH 6.0). Endogenous peroxidase was inhibited by 0.3% H2O2, and non-specific protein binding was blocked with 10% goat serum. The sections were then incubated with primary antibody against AFP and CXCR4 (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Non-immune immunoglobulin G(IgG) was used as a negative control, and antigenic sites were localized using a SP9000 Polymer Detection System and a 3,3′-diaminobenzidine kit (ZSGB-BIO, Beijing, China).
Sp9000 polymer detection system
Manufactured by ZSGB-BIO
Sourced in China
The SP9000 Polymer Detection System is a laboratory instrument designed to detect and analyze polymers. It utilizes advanced technology to identify and characterize various polymer samples. The core function of the SP9000 is to provide accurate and reliable polymer analysis for research and testing purposes.
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7 protocols using sp9000 polymer detection system
1
Immunohistochemical Analysis of AFP and CXCR4
2
Immunohistochemical Analysis of AFP and CXCR4 in HCC
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The expression and cellular distribution of AFP and CXCR4 proteins in HCC specimens were assessed by immunohistochemical analysis. Five‐millimetre‐thick paraffin sections were deparaffinized and rehydrated according to standard protocols, and heat‐induced antigen retrieval was performed in sodium citrate buffer (10 mmol/l, pH 6.0). Endogenous peroxidase was inhibited by 0.3% H2O2, and non‐specific protein binding was blocked with 10% goat serum. The sections were then incubated with primary antibody against AFP and CXCR4 (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Non‐immune immunoglobulin G was used as a negative control, and antigenic sites were localized using a SP9000 Polymer Detection System and a 3,3′‐diaminobenzidine kit (ZSGB‐BIO, Beijing, China).
3
Immunohistochemical Evaluation of Tumor Markers
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The protein expression of c-myc, Ras, activated caspase-3 and PARP-1 in tumor-bearing mice were evaluated by immunohistochemical analysis. Briefly, tumorous tissue was removed from tumor-bearing mice and cut into 5-mm-thick paraffin sections. The sections were deparaffinized and rehydrated according to standard protocols. The sections were then incubated with primary antibody (1:100 dilution; Abcam Trading Company, Ltd. Shanghai, China) at 4 °C overnight. Nonimmune (IgG) was used as a negative control, and antigenic sites were identified using an SP9000 Polymer Detection System and a 3,3’-diaminobenzidine kit (ZSGB-BIO, Beijing, China). The standard protocols were performed in accordance with approved guidelines [19 (link), 20 (link)].
4
Immunohistochemical Analysis of HCC Markers
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The expression and cellular distribution of AFP, Ras and CXCR4 proteins in HCC specimens were assessed by immunohistochemical analysis. Five-millimeter-thick paraffin sections were deparaffinized and re-hydrated according to standard protocols, and heat-induced antigen retrieval was performed in sodium citrate buffer (10 mmol/L, pH 6.0). Endogenous peroxidase was inhibited by 0.3% H2O2, and non-specific protein binding was blocked with 10% goat serum. The sections were then incubated with primary antibody against AFP, Ras and CXCR4 (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Non-immune immunoglobulin G(IgG) was used as a negative control, and antigenic sites were localized using a SP9000 Polymer Detection System and a 3,3′-diaminobenzidine kit (ZSGB-BIO, Beijing, China).
5
Immunohistochemical Analysis of Stemness Markers in HCC
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The expression and cellular distribution of GATA5, β‐catenin, p‐Oct4 (Thr235), Nanog, Klf4, c‐myc and EpCAM proteins in HCC specimens were assessed by immunohistochemical analysis. Five‐millimetre‐thick paraffin sections were deparaffinized and rehydrated according to standard protocols, and heat‐induced antigen retrieval was performed in a sodium citrate buffer (10 mmol/L, pH 6.0). Endogenous peroxidase was inhibited by 0.3% H2O2, and non‐specific protein binding was blocked with 10% goat serum. The sections were then incubated with primary antibody against GATA5, β‐catenin, p‐Oct4 (Thr235), Nanog, Klf4, c‐myc and EpCAM (1:100 dilution; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) at 4°C overnight. Non‐immune immunoglobulin G (IgG) was used as a negative control, and antigenic sites were localized using an SP9000 Polymer Detection System and a 3,3′‐diaminobenzidine kit (ZSGB‐BIO, Beijing, China). Methods were performed in accordance with the approved guidelines.14
6
Evaluating Apoptotic Markers in Tumor Mice
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The protein expression of C-myc, Ras, activated caspase-3 and PARP-1 in tumor-bearing mice was evaluated by immunohistochemical analysis. Brie y, tumorous tissue was removed from tumor-bearing mice and cut into ve-millimeter-thick para n sections. The sections were depara nized and rehydrated according to standard protocols. The sections were then incubated with primary antibody(1:100 dilution; Abcam Trading (Shanghai) Company, Ltd., Shanghai, China) at 4 °C overnight. Nonimmune (IgG) was used as a negative control, and antigenic sites were identi ed using an SP9000 Polymer Detection System and a 3,3′-diaminobenzidine kit (ZSGB-BIO, Beijing, China). The standard protocols were performed in accordance with approved guidelines [14, 17] .
Analysis of caspase-3 activity HLE, Bel 7402 and PLC/PRF/5 cells were treated with tumor necrosis factor-related apoptosis-induced ligand (TRAIL) (2 μmol/L) or Vinco (80 μg/ml) and a caspase-3 inhibitor (Z-DEVD-FMK, 1.0 μmol/L) (Selleck Chemicals Company, USA) for 24 h. Caspase-3 activity was measured with a commercial kit (APOP-CYTO Caspase-3 Colorimetric Assay Kit; Medical and Biological Laboratories, Japan) according to the manufacturer's protocol as described in a previous study [18] .
Analysis of caspase-3 activity HLE, Bel 7402 and PLC/PRF/5 cells were treated with tumor necrosis factor-related apoptosis-induced ligand (TRAIL) (2 μmol/L) or Vinco (80 μg/ml) and a caspase-3 inhibitor (Z-DEVD-FMK, 1.0 μmol/L) (Selleck Chemicals Company, USA) for 24 h. Caspase-3 activity was measured with a commercial kit (APOP-CYTO Caspase-3 Colorimetric Assay Kit; Medical and Biological Laboratories, Japan) according to the manufacturer's protocol as described in a previous study [18] .
7
Evaluating Apoptotic Markers in Tumor Mice
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The protein expression of c-myc, Ras, activated caspase-3 and PARP-1 in tumor-bearing mice were evaluated by immunohistochemical analysis. Brie y, tumorous tissue was removed from tumor-bearing mice and cut into ve-millimeter-thick para n sections. The sections were depara nized and rehydrated according to standard protocols. The sections were then incubated with primary antibody(1:100 dilution; Abcam Trading Company, Ltd. Shanghai, China) at 4 °C overnight. Nonimmune(IgG) was used as a negative control, and antigenic sites were identi ed using an SP9000 Polymer Detection System and a 3,3′-diaminobenzidine Kit(ZSGB-BIO, Beijing, China). The standard protocols were performed in accordance with approved guidelines [Li et al., 2013; Lu et al., 2016] .
Analysis Of Caspase3 Activity HLE, Bel 7402 and PLC/PRF/5 cells were treated with tumor necrosis factor-related apoptosis-induced ligand(TRAIL) (2 µmol/L) or Vinco(80 µg/ml) and a caspase-3 inhibitor(Z-DEVD-FMK, 1.0 µmol/L) (Selleck Chemicals Company, USA) for 24 h. Caspase-3 activity was measured with a commercial Kit(APOP-CYTO Caspase-3 Colorimetric Assay Kit; Medical and Biological Laboratories, Japan) according to the manufacturer's protocol as described in a previous study [Li et al., 2009] .
Analysis Of Caspase3 Activity HLE, Bel 7402 and PLC/PRF/5 cells were treated with tumor necrosis factor-related apoptosis-induced ligand(TRAIL) (2 µmol/L) or Vinco(80 µg/ml) and a caspase-3 inhibitor(Z-DEVD-FMK, 1.0 µmol/L) (Selleck Chemicals Company, USA) for 24 h. Caspase-3 activity was measured with a commercial Kit(APOP-CYTO Caspase-3 Colorimetric Assay Kit; Medical and Biological Laboratories, Japan) according to the manufacturer's protocol as described in a previous study [Li et al., 2009] .
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