For immunohistochemistry, 4-µm-thick tumor sections were immersed in 0.3% hydrogen peroxide for 10 min, microwaved in citrate phosphate buffer (pH 6.0) and incubated with 10% normal goat serum (Maixin Biotech. Co., Ltd., Fuzhou, China) for 30 min. The tumor slices were incubated overnight at 4°C with the following primary monoclonal antibodies (Maixin Biotech. Co., Ltd.): Rabbit anti-COX-2 monoclonal antibodies (catalog no., RMA-0549); mouse anti-MMP-2 monoclonal antibodies (catalog no., MAB-0244); and mouse anti-VEGF monoclonal antibodies (catalog no., MAB-0243). Secondary anti-mouse/rabbit antibodies from Kit-5030 (Maixin Biotech. Co., Ltd.) were then incubated with the samples at 37°C for 15 min. Primary and secondary antibodies were working solution and were not diluted. A standard staining procedure was finished by using a 3,3′-diaminobenzidine kit (Kit-0014; Maixin Biotech. Co., Ltd.). Immunostaining was evaluated blindly by a board-certified pathologist (Capital Medical University), who assigned the intensity and prevalence score as described previously (25 (link)). Five microscopic fields were randomly selected and viewed at a magnification of ×100. Briefly, the intensity of staining was scored as follows: 0, negative; 1, weak staining; 2, moderate staining; and 3, strong staining.
Normal goat serum (ngs)
Manufactured by Maixin Group
Sourced in China
Normal goat serum is a laboratory reagent derived from the blood of healthy goats. It is typically used as a blocking agent or diluent in various immunological and biochemical assays to reduce non-specific binding and background signals.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Desmin, by Cell Signaling Technology (1 mentions)
Anti nestin, by R&D Systems (1 mentions)
βiii tubulin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
3 3 diaminobenzidine (dab), by Maixin Group (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
4 protocols using normal goat serum (ngs)
1
Immunohistochemistry of Tumor Markers: COX-2, MMP-2, and VEGF
2
Differentiation of hHF-MSC-derived iPSCs
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hHF-MSC-derived iPSCs were harvested by collagenase type IV. After settling, the supernatant was aspirated and the MEF medium was replaced to remove the MEF. hHF-MSC-derived iPSCs were transferred to petri dishes in the MEF medium. After an 8 days floating culture, embryoid bodies were transferred to gelatin-coated plates and were then incubated for another 16 days. After the incubation, the cells were fixed with 4% paraformaldehyde in PBS and then incubated in PBS containing 5% normal goat serum (Maixin Biotech, Fuzhou, China), 1% bovine serum albumin (BSA, Biotopped, China), and 0.2% Triton X-100. The primary antibodies were as follows: anti-alpha smooth muscle actin (α-SMA, R&D, USA), anti-alpha fetoprotein polyclonal antibody (AFP, R&D, USA), and anti-Nestin (R&D, USA). vimentin(Cell Signaling, USA), desmin (Cell Signaling, USA), βIII-tubulin (Cell Signaling, USA). The secondary antibodies were Alexa 555-labeled anti-mouse IgG (1:1000, Cell Signaling, USA). Nuclei were stained with 1 mg/ml Hoechst 33342 (Invitrogen, USA).
3
Immunohistochemical Detection of Bak Protein
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Slides were deparaffinized with xylene twice (each for 10 min) and hydrated through an ethanol gradient (100%, 95%, 90%, 80% and 70%, each for 5 min), and rinsed with phosphate-buffered saline (PBS). Bak antigen was retrieved by citric acid buffer (pH6.0) microwave antigen retrieval (high power for 3 min and low power for 15 min).The slides then were treated with 3% H2O2 for 15 min and washed by tris-buffered saline containing 1% Tween 20 (TBST) for 15 min. Following a 30 min blocking with normal goat serum (Maixin, Fujian, China), slides were incubated with mouse monoclonal anti-Bak (Cell Signaling Technology, Danvers, MA; 1:500 dilution) overnight at 4°C. On the next day, slides were washed three times with TBST (each for 15 min). Slides were then incubated with a secondary anti-mouse antibody (Maixin, Fujian, China) for 90 min at 37°C and washed twice with TBST (each for 15 min). Then the slides were visualized with 3,3’-diaminobenzidine (DAB) (Maixin, Fujian, China) for 5 min and counterstained with haematoxylin for 90 seconds. The slides were mounted and photographed with Olympus BX51 microscope (Olympus, Japan).
4
Immunofluorescence Staining of Decidual Stromal Cells
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The DPCs were fixed in 4% paraformaldehyde (Ding Guo, Beijing, China) for 10 min and permeabilized with 0.1% Triton X-100 (Sigma) for 20 min at room temperature. Normal goat serum (10%; Maixin, Fuzhou, China) was used to block nonspecific binding. The DPCs were then incubated overnight with primary antibodies at 4°C (detailed information regarding the primary antibodies is listed in Table 2 ), followed by incubation with Alexa Fluor 488/555-conjugated secondary antibodies (Cell Signaling Technology, Danvers, USA) for 1 h at room temperature. Unbound antibody was removed by thoroughly washing with PBS. Nuclei were stained with Hoechst 33342 (Invitrogen). For the negative control, primary antibodies were omitted.
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