TMA sections (5 μm) were deparaffinized, hydrated, blocked for endogenous peroxidases and antigen retrieval. After blocking for an hour at room temperature, the slide was incubated with primary antibody at 4°C overnight. And then samples were probed with biotinylated goat anti-rabbit secondary antibody. Finally, the slide was detected by SignalStain® DAB (Cell Signaling Technology, Danvers, MA) and counterstained with haematoxylin QS (Vector Laboratories). Cells containing brown granules were independently counted by two pathologists who were blinded to clinical parameters, and the samples were scored according to the proportion of positive cells as follows: 0, none; 1, <25%; 2, 25%-50%; 3, 51%-75%; and 4, 76%-100%. The staining intensity was scored as follows: 0, none; 1, weak; 2, moderate; and 3, strong. The total staining score (range 0-12) was calculated by multiplying the two subscores, and the samples with scores of 0-3, 4-6 and 9-12 were respectively classified as low expression, moderate expression and high expression.
Signalstain dab
Manufactured by Cell Signaling Technology
Sourced in United States
SignalStain® DAB is a high-quality immunohistochemistry detection reagent. It is designed for the visualization of target proteins in tissue sections or cell preparations.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin qs, by Vector Laboratories (6 mentions)
Vectamount aq, by Vector Laboratories (4 mentions)
Signalstain boost detection reagent, by Cell Signaling Technology (4 mentions)
Haematoxylin qs, by Vector Laboratories (2 mentions)
Hematoxylin, by Cell Signaling Technology (2 mentions)
Animal free blocking solution, by Cell Signaling Technology (2 mentions)
Anti tgf β1 receptor, by Abcam (2 mentions)
Signalstain boost detection reagent hrp mouse, by Cell Signaling Technology (2 mentions)
Anti cd8 clone c8 144b, by Agilent Technologies (1 mentions)
Signalstain ab diluent, by Cell Signaling Technology (1 mentions)
Immunohistochemistry protocol, by Cell Signaling Technology (1 mentions)
Anti mpo, by Cell Signaling Technology (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Blocking solution, by Cell Signaling Technology (1 mentions)
Immunohistochemistry protocol paraffin, by Cell Signaling Technology (1 mentions)
Rabbit anti human eno1 antibody, by Proteintech (1 mentions)
Anti tgf β1, by Santa Cruz Biotechnology (1 mentions)
16 protocols using signalstain dab
1
Immunohistochemical Analysis of Protein Expression
2
Immunohistochemical Analysis of PD-L1 and CD8+ T Cells
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After formalin-fixed and paraffin-embedded tissue samples of 4 μm-thick sections were treated with xylene and ethanol, antigen retrieval was performed in ethylene glycol and sodium azide, pH 9.0, at 120°C for 20 minutes. The sections were then incubated with anti-PD-L1 (clone E3L1N; Cell Signaling Technology) at a 1:100 dilution and with anti-CD8 (clone C8/144B; Dako, Glostrup, Denmark) at a 1:400 dilution overnight at 4°C. Subsequently, the sections were treated with secondary antibody at room temperature for 30 minutes. The slides were incubated with the SignalStain DAB (Cell Signaling Technology) and counterstained with hematoxylin. They were observed under the microscope (Keyence, Osaka, Japan), and the images were collected. Tumor PD-L1 expression was defined as positive when staining of the tumor-cell membrane (at any intensity) was observed in ≥1% of the cells, and as negative when no staining or positive staining was detected in <1% of the cells. We set a threshold of 1% based on a previous phase III trial involving an anti-PD-1 agent and another study [27 (link)28 (link)]. CD8+ lymphocyte infiltration was defined as positive when there were ≥30 CD8+ lymphocytes in a 40× field of view. Two independent investigators assessed the expressions and the infiltrations.
3
Immunohistochemical Staining of Tissue Sections
4
Immunohistochemical Assay for CDK9 Expression
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The expression of CDK9 was determined using IHC assays according to the manufacturer's instructions (Cell Signaling Technology, Beverly, MA, USA). In brief, the paraffin-embedded slides were baked for 1 h at 60 °C before xylene deparaffinization and subsequent rehydration through graded ethanol (100% and 95%). 3% hydrogen peroxide was used to quench endogenous peroxidase activity after heated epitope retrieval. Following this, the slide was blocked for 1 h with normal goat serum, and then incubated with polyclonal rabbit antibody to human CDK9 (Cell Signaling Technology, catalog #2316S, 1:50 dilution, in 1% bovine serum albumin PBS) overnight in a humidified chamber set at 4 °C. SignalStain® Boost Detection Reagent (Cell Signaling Technology) and SignalStain® DAB (Cell Signaling Technology) were then utilized to detect the bound antibody. A hematoxylin QS (Vector Laboratories, Burlingame, CA) counterstain was used to obtain clearer images of the osteosarcoma cell nuclei before final long-term preservation using VectaMount AQ (Vector Laboratories) section mounting. Even in the absence of CDK4 antibody binding, the TMA slides were stained to reveal any nonspecific secondary antibody reactions.
5
Immunohistochemical Staining of Tissue Sections
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The tissue sections were deparaffinized with xylene and rehydrated in gradient ethanol. After repairing with 1× citrate, the parts were placed in a 3% aqueous hydrogen peroxide solution and incubated for 10 minutes to remove endogenous peroxidase. Next, the sections were blocked with a blocking solution for 1 hour at room temperature and then incubated with the primary antibody (#9188, 1:125, Cell Signaling Technology, USA) at 4 °C overnight. After washing with TBST, the sections were incubated with the secondary antibody (#8114, Cell Signaling Technology, USA) for 30 minutes at room temperature, and stained with SignalStain® DAB. The images were seen under a light microscope.
6
Immunohistochemical Analysis of CD44 Expression
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The expression levels of CD44 were evaluated following the Immunohistochemistry Protocol (Cell Signaling Technology, Beverly, MA). In brief, 5-μm paraffin tissue section slides were baked for 1 hour at 60 °C, deparaffinized in xylene three times for 10 minutes each, and then transferred through graded ethanol (100%, 95%) for rehydration. After antigen retrieval, 3% hydrogen peroxide was applied to quench the endogenous peroxidase for 10 minutes. Following protein blocking for 1 hour at room temperature, the slide was incubated with primary antibody (dilution 1:50) at 4 °C overnight in a humidified chamber. Subsequently, bound antibody on the array was detected by SignalStain® Boost Detection Reagent (Cell Signaling Technology) and SignalStain® DAB (Cell Signaling Technology). To obtain better images and long-term preservation, the section was counterstained with hematoxylin QS (Vector Laboratories) and mounted with VectaMount AQ (Vector Laboratories), respectively. The immunostaining intensity of CD44 was assessed independently by two scientists who had no knowledge of the clinical information, as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; and 3+, intense staining. The negative and positive staining controls are shown in Supplementary data Figure S1 ).
7
Immunohistochemical Analysis of MPO
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The lung tissues of mice were incubated in 4% paraformaldehyde for fixation and paraffin embedding. After dewaxing in xylene, paraffin-embedded sections were dehydrated in graded ethanol. Incubation for 30 min with 0.3% H2O2 in PBS was followed by blocking with PBS containing 1% BSA for 1 h. Proteins of interest were recognized by incubation with anti-MPO (Cell Signaling, 1:1000) at 4°C overnight. After washing with TBST, sections were incubated at room temperature for 30 min with SignalStain Boost IHC Detection Reagent (CST, United States). SignalStain DAB (Cell Signaling Technology) was then applied to the sections to reveal the intensity of the staining. Sections were captured using an Olympus BX-51 microscope (Olympus, Japan).
8
Immunohistochemical Analysis of TGF-β1 and Receptor
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To examine protein localization and expression, the formalin-fixed tissues were de-paraffinized. After deparaffinization and dehydration of paraffin sections, heat-induced epitope retrieval was performed using a pressure cooker and sodium citrate buffer (10 mM sodium citrate, 0.05% tween-20, pH 6.0). Once boiled, slides were transferred from PBS to the sodium citrate buffer in pressure cooker for 10 min. Slides were then cooled to room temperature for 30 min, and permeabilized with permeabilization buffer containing 0.3% triton-100 in PBS for 10 min. To block endogenous peroxidase activity, slides were incubated in 3% hydrogen peroxide for 10 min and blocked with animal-free blocking solution (Cat# 15019; Cell Signaling, Inc.). After blocking, slides were incubated overnight at 4°C with mouse monoclonal anti-TGF-β1 (1:50; Cat# sc-130348; Santa Cruz, Inc.) or rabbit polyclonal anti-TGF-β1 receptor (1:100; Cat# ab235178; Abcam, Inc.) and subsequently incubated with SignalStain Boost Detection Reagent (HRP mouse; Cat# 8125; or HRP Rabbit; Cat# 8114; Cell Signaling, Inc.) for 30 min at room temperature. Slides were then incubated for 2–10 min with SignalStain DAB (Cat# 8059; Cell Signaling, Inc.), immersed in distilled H2O, stained with hematoxylin (Cat# 14166; Cell Signaling, Inc.) and mounted with coverslips. All washing steps were done three times with PBS-T (tween-20, 0.05%).
9
Immunohistochemical Analysis of Pgp and CD44 in Ovarian Cancer
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Immunohistochemistry was performed in an ovarian cancer tissue microarray (TMA)54 (link) and in mice tumor tissues by using an Immunohistochemistry Protocol (Paraffin) and reagents from Cell Signaling Technology to detect the expression level of Pgp and CD44. Briefly, paraffin tissue sections from excised tumors were deparaffinized and hydrated through xylene and graded alcohol. Antigens were unmasked by heat treatment and endogenous peroxidase was inhibited by incubating with 3% H2O2 for 10 min. After blocking each section with blocking solution (Cell Signaling Technology) for 1 h at room temperature, they were incubated with primary antibody in a humidified chamber at 4°C overnight. Sections were then washed with TBST, and the signal were developed using SignalStain® Boost Detection Reagent (Cell Signaling Technology) and SignalStain® DAB (Cell Signaling Technology). Finally, the slides were mounted with VectaMount AQ (Vector Laboratories).
10
Tissue Microarray Analysis of Liver Cancer
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The tissue microarray (TMA), including 30 paired liver cancer tissues and para-carcinoma tissues, was derived from 2019 to 2021 at the First Affiliated Hospital of Wenzhou University. This study was supported by the hospital’s ethics committee, and all the patients provided informed consent.
TMA sections (4-μm thick) were deparaffinized and hydrated, and 0.3% hydrogen peroxide, and incubated with primary antibody overnight at 4°C and with secondary biotinylated goat anti-rabbit antibody successively; the sections were then stained using SignalStain® DAB (Cell Signaling Technology, Danvers, MA) and counterstained with hematoxylin QS (Vector Laboratories). The intensity of staining (0, 1, 2, 3) and the proportion of positive cells (0%–100%) were semi-quantified, and scored from 0 (no stained cells) to 3 (all cells intensely stained). The detail information of tissue microarrays is available inSupplementary Table 1
TMA sections (4-μm thick) were deparaffinized and hydrated, and 0.3% hydrogen peroxide, and incubated with primary antibody overnight at 4°C and with secondary biotinylated goat anti-rabbit antibody successively; the sections were then stained using SignalStain® DAB (Cell Signaling Technology, Danvers, MA) and counterstained with hematoxylin QS (Vector Laboratories). The intensity of staining (0, 1, 2, 3) and the proportion of positive cells (0%–100%) were semi-quantified, and scored from 0 (no stained cells) to 3 (all cells intensely stained). The detail information of tissue microarrays is available in
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