Immunohistochemistry assay was performed by using antibody PECAM-1. Staining was performed with DAB Kit (Sangon Biotech; Shanghai) according to the manufacturer's instructions.
Dab kit
Manufactured by Sangon
Sourced in China
The DAB kit is a laboratory reagent used for the detection and visualization of target proteins in biological samples. It provides a chromogenic reaction that produces a brown-colored precipitate at the site of the target protein, allowing for its identification and localization.
Automatically generated - may contain errors
Lab products found in correlation
Ultrasensitive s p detection kit, by Maixin Group (4 mentions)
β catenin, by Abcam (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Ab32572, by Abcam (1 mentions)
Anti col1, by Abcam (1 mentions)
Anti col 3, by Abcam (1 mentions)
Anti α sma, by Abcam (1 mentions)
Hematoxylin, by Sangon (1 mentions)
Anti his mouse monoclonal antibody, by Transgene (1 mentions)
Coomassie brilliant blue r 250, by Sangon (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Tak 242, by MedChemExpress (1 mentions)
Rabbit anti lc3 polyclonal antibody, by Abcam (1 mentions)
11 protocols using dab kit
1
PECAM-1 Immunohistochemistry Assay
2
Immunohistochemistry of Tumor Angiogenesis
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Paraffin-embedded tumour sections were placed on adherent slides, then deparaffinized in xylene, and further dehydrated with different concentrations of alcohol, and then pretreated in citrate buffer for 20 min in a 98 °C steamer, for antigen retrieval. Sections were incubated overnight at 4 °C with antibodies against CD34, NRP1 or VEGFR1/2. The UltraSensitive S-P Detection Kit (KIT-9720, Maixin, Fuzhou, China) was used to perform immunostaining, and the DAB kit (PW017, Sangon Biotech, Shanghai, China) was used to develop the colour. Subsequently, the haematoxylin was used to counterstain sections. Morphological assessment was finally performed using H&E staining.
3
Immunohistochemical Analysis of ITCH and β-Catenin
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Paraffin-embedded blocks were sectioned onto positively charged microscope slides and then were deparaffinized with xylene. After deparaffinization, the slides were hydrated with absolute ethanol and pretreated with citrate buffer for 20 min in a 98 °C steamer to retrieve the antigens. Then, the slides were incubated with antibodies against ITCH (1:200, Abcam, Cambridge, UK) or β-catenin (1:250, Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, an UltraSensitive S-P Detection kit (KIT-9720, Maixin, Fuzhou, China) was used to perform immunostaining, and a DAB kit (PW017, Sangon Biotech, Shanghai, China) was used to develop colour. Finally, the samples were counterstained with haematoxylin, after which the integrated optical density (IOD/Area) in different groups were examined using Image-Pro Plus 6.0.
4
Immunostaining of Tumor Sections
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Tumors embedded in paraffin were sectioned onto positively charged microscope slides. The sections were deparaffinized in xylene, hydrated in graded alcohol, and pretreated for antigen retrieval in citrate buffer for 20 min in a 98°C steamer(Bmi-1 and Brdu). The sections were incubated at 4°C overnight with Brdu (1:200, Maixin, Fuzhou, China) and Bmi-1 (1:100BD Biosciences USA). Immunostaining was performed using an UltraSensitive S-P Detection Kit (KIT-9720, Maixin, Fuzhou, China), and the color was developed using a DAB kit (PW017, Sangon Biotech, Shanghai, China). A TUNEL assay was performed with an In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions.
5
Immunohistochemical Analysis of ECT2 in Osteosarcoma
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Biopsied osteosarcoma tissues before chemotherapy treatment were collected and embedded in paraffin. The detailed procedures for IHC staining have been described previously [25 (link)]. Briefly, tissue slides were treated as followed: de-waxed, rehydrated, antigen retrieval, goat serum blocking, primary antibody incubation (ECT2, Cat. #ab151503, Abcam, Cambridge, UK), secondary antibody incubation, and immunostaining using the DAB kit (Cat. #PW017, Sangon Biotech, Shanghai, China). Nonimmune serum was used as negative control.
The expression level of ECT2 protein was semi-quantified from the immunostaining results by two independent pathologists. The percentage of positive stained cells was calculated and scored as 0 (0–5% positive), 1 (5–25% positive), 2 (26–50% positive) and 3 (51–100% positive). The staining intensity was also scored as 0 (negative), 1 (weak, pale yellow), 2 (moderate, dark yellow), and 3 (strong, brown). The final immunoreactivity score (IRS) was calculated by multiplying the percentage and intensity scores (ranging from 0–9). To explore the role of ECT2 in osteosarcoma, patients were classified into two groups based on the IRS: low expression (IRS 0–4) and high expression (IRS 5–9).
The expression level of ECT2 protein was semi-quantified from the immunostaining results by two independent pathologists. The percentage of positive stained cells was calculated and scored as 0 (0–5% positive), 1 (5–25% positive), 2 (26–50% positive) and 3 (51–100% positive). The staining intensity was also scored as 0 (negative), 1 (weak, pale yellow), 2 (moderate, dark yellow), and 3 (strong, brown). The final immunoreactivity score (IRS) was calculated by multiplying the percentage and intensity scores (ranging from 0–9). To explore the role of ECT2 in osteosarcoma, patients were classified into two groups based on the IRS: low expression (IRS 0–4) and high expression (IRS 5–9).
6
Immunohistochemical Detection of β-Catenin and CTHRC1
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After dewaxing, hydration, washing, endogenous peroxidase neutralization and antigen recovery, the 4 μm-thick tissue sections were blocked with goat serum at 37°C for 1 h, and then incubated overnight with anti-β-catenin (ab32572, 1:500, Abcam, Cambridge, UK) and anti-CTHRC1 antibodies (16534-1-AP, 1:20, Proteintech, Chicago, IL, USA) at 4°C. On the second day, the sections were washed with phosphate buffered saline (PBS) and processed with a two-step assay kit (PV-9000, GBI, USA). Subsequently, the sections were stained with DAB kit (PW017, Sangon Biotech, Shanghai, China) and hematoxylin, dried at 65°C, and sealed, followed by observation using an optical microscope (CX23, Olympus, Japan).
7
Immunohistochemical Analysis of Kidney Fibrosis
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Immunohistochemistry experiments were conducted on paraffin-embedded kidney tissue sections using anti-CoL I, anti-CoL III, and anti-α-SMA antibodies. The kidney tissue sections were dewaxed and dehydrated and then put in citrate buffer at 95 °C for 10–15 min, and then the tissue sections were incubated with anti-CoL I (Abcam), anti-CoL III (Abcam) and anti-α-SMA (Abcam) antibodies at 4 °C for about 12 h. The sections were incubated with the secondary antibody (Abcam). The DAB kit (Sangon Biotech) was applied to visualize the slices and counterstained the slices with hematoxylin (Sangon Biotech).
8
Purification and Western Blot of His-tagged Proteins
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The cells containing recombinant proteins were centrifuged at 12,000 rpm for 1 min. After adding 24 μL PBS and 6 μL 5X protein loading buffer, the precipitates were placed in boiling water for 10 min before being stored in ice. Subsequently, 10 μL of the protein solution was used for 12% polyacrylamide gel electrophoresis (PAGE) and dyed with Coomassie brilliant blue R250 (Sangon), or used for western blot analysis.
Western blot analysis was initiated by transferring the proteins from the PAGE gels to polyvinylidene difluoride films (Millipore, USA). The films were washed with PBS with Tween ® 20 (PBST) three times, each time for 10 min. A non-specific antibody recognized sites on the films blocked with 5% fat-free milk powder, and the films were then incubated with mouse anti-His monoclonal antibody (Transgen) for 1 h at room temperature. The films were washed with PBST another three times, and then incubated with HRP-labeled goat anti-mouse polyclonal antibody (Transgen). After again being washed three times with PBST, the films were colored using a DAB kit (Sangon).
Western blot analysis was initiated by transferring the proteins from the PAGE gels to polyvinylidene difluoride films (Millipore, USA). The films were washed with PBS with Tween ® 20 (PBST) three times, each time for 10 min. A non-specific antibody recognized sites on the films blocked with 5% fat-free milk powder, and the films were then incubated with mouse anti-His monoclonal antibody (Transgen) for 1 h at room temperature. The films were washed with PBST another three times, and then incubated with HRP-labeled goat anti-mouse polyclonal antibody (Transgen). After again being washed three times with PBST, the films were colored using a DAB kit (Sangon).
9
LPS-Induced Inflammation Model in Rats
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LPS was purchased from Sigma-Aldrich (Darmstadt, Germany). The cigarettes were from Liuzhou Cigarette Factory (Liuzhou, China). Gentamicin (cat. no. 15710-064) was from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Rat monoclonal antibody against TLR4 was from Abcam (Cambridge, MA, USA). Horseradish peroxidase-conjugated anti-rat IgG was from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). The DAB kit was from Sangon Biotech Co., Ltd. (Shanghai, China). TAK-242 was from MedChem Express (Monmouth Junction, NJ, USA). The ELISA kit (cat. no. E-EL-R0009c) was from R&D Systems, Inc. (Minneapolis, MN, USA).
10
Immunohistochemical Analysis of Tumor Angiogenesis
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Tumors preserved in formalin were placed in paraffin blocks and sectioned onto positively charged microscope slides. The sections were deparaffinized in xylene, hydrated in graded alcohol, and pretreated for antigen retrieval in citrate buffer for 20 min in a 98°C steamer (CD34 and NRP1). The sections were incubated at 4°C overnight with CD34 (1∶200, Santa Cruz Biotechnology) and NRP1 (1∶100 R&D Systems). Immunostaining was performed using the UltraSensitive S-P Detection Kit (KIT-9720, Maixin, Fuzhou, China), and the color was developed using a DAB kit (PW017, Sangon Biotech, Shanghai, China).Subsequently, the sections were counterstained with hematoxylin. The slides were then stained with H&E to assess morphology.
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