Sections were deparaffinized and rehydrated before undergoing heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0, Solarbio, cat. C1013) for 30 minutes. Slides were blocked for endogenous peroxidase for 20 minutes, then blocked for 1 hour in 5% BSA, 1% goat serum, 0.1% Tween-20 buffer in PBS, and incubated overnight at 4°C with the primary antibody diluted in blocking buffer or SignalStain® Antibody Diluent (cat. 8112, Cell Signaling Technology). Slides were incubated with biotinylated secondary antibodies for 1 hour at room temperature, and the signal was detected using the Vectastain Elite ABC kit (cat. PK-6105, Vector Laboratories, Newark, CA, USA). Hematoxylin was used for counterstaining in IHC. Antibodies and dilutions used in the studies: Ki67 (cat. 12202, 1:500, Cell Signaling Technology), Desmin (cat. 5332, 1:100, Cell Signaling Technology).
Signalstain antibody diluent
Manufactured by Cell Signaling Technology
Sourced in United States
SignalStain Antibody Diluent is a ready-to-use solution designed to dilute primary antibodies for immunohistochemistry and western blotting applications. It is a buffered solution that helps maintain antibody stability and performance during the staining process.
Automatically generated - may contain errors
Lab products found in correlation
Signalstain boost detection reagent, by Cell Signaling Technology (3 mentions)
Vectastain elite abc kit, by Vector Laboratories (3 mentions)
Immpress reagent kit, by Vector Laboratories (3 mentions)
Immpact dab peroxidase substrate, by Vector Laboratories (3 mentions)
Discovery ultra system, by Roche (3 mentions)
Signalstain boost ihc detection reagent, by Cell Signaling Technology (3 mentions)
Axio observer, by Zeiss (2 mentions)
Dp21 camera, by Olympus (2 mentions)
Anti trimethyl histone h3 lys27 antibody, by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Signalstain citrate unmasking solution, by Cell Signaling Technology (2 mentions)
Signalstain dab substrate kit, by Cell Signaling Technology (2 mentions)
Evos microscope, by Thermo Fisher Scientific (2 mentions)
Desmin, by Cell Signaling Technology (1 mentions)
Primary ki 67 antibody, by Cell Signaling Technology (1 mentions)
Xylene, by Fujifilm (1 mentions)
Goat serum, by Merck Group (1 mentions)
Mayer s hematoxylin solution, by Fujifilm (1 mentions)
Anti cd68 antibody, by Cell Signaling Technology (1 mentions)
Eosin y solution, by Fujifilm (1 mentions)
35 protocols using signalstain antibody diluent
1
Immunohistochemical Analysis of Ki67 and Desmin
2
Immunohistochemical Staining of Ki-67 in Paraffin Sections
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Paraffin embedded sections were deparaffinized and rehydrated through a xylene, 100% ETOH, 95% ETOH, dH2O gradient. Slides were then washed in dH2O and antigen was unmasked in boiling 10mM sodium citrate pH 6.0 for 10 minutes. Slides were incubated in 3% hydrogen peroxide and each slide was blocked in 5% Goat Serum/TBST for one hour. Primary Ki-67 antibody (CS-12202) Cell Signaling Technology (Beverly, MA) was diluted 1:400 in SignalStain Antibody Diluent Cell Signaling Technology (Beverly, MA) and incubated overnight at 4°C. Slides were washed with TBST and covered with three drops of SignalStain Boost Detection Reagent (CS-8114) Cell Signaling Technology (Beverly, MA). After washing with TBST slides were stained with SignalStain DAB Substrate kit (CS-8059) Cell Signaling Technolgy (Beverly, MA), counterstained with hematoxylin, dehydrated, and coverslipped.
3
Immunohistochemical Staining Protocol
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An anti-CD68 antibody, 1× Tris-buffered saline with Tween® 20, SignalStain® antibody diluent, SignalStain® Boost IHC detection reagents, and SignalStain® DAB substrate kits were purchased from Cell Signaling Technology (Danvers, MA, USA). Xylene, mounting medium, Mayer’s hematoxylin solution, and 1% Eosin Y solution were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Goat serum was purchased from Merck (Darmstadt, Germany).
4
Quantifying EGFR Expression in Erlotinib-Resistant Tumors
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Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded WHIM2 tumors. Heat-induced antigen retrieval was performed in pH 9 Tris–EDTA using a Dakocytomatin Pascal Pressure Chamber. EGFR (Cell Signaling Technology, 2232S) antibody was diluted 1:50 in SignalStain Antibody Diluent (Cell Signaling Technology) and was applied to sections from parental and erlotinib-resistant WHIM2 tumors. Detection was performed using the Rabbit Dako EnVision System (Agilent K406511–2). Slides were imaged using Zeiss Axio Observer with Zen software. Quantification of immunohistochemical staining was performed using ImageJ plugins IHC Toolbox and IHC Profiler28 (link). T-tests were performed on GraphPad Prism v.9.2.0 to determine if differences in staining were statistically significant between groups.
5
Acetylation Dynamics in Pancreatic Cancer
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A total of 102 patients who underwent curative operation for pancreatic cancer at Tokyo Medical and Dental University Hospital between 2007 and 2016. With approval of the ethics committees of the Faculty of Medicine in Tokyo Medical and Dental University (permission No. M2000-1080, G2017-018), written informed consent was obtained from all patients. Patients were anonymously coded in accordance with ethical guidelines, as instructed by the Declaration of Helsinki.
Heat-induced epitope retrieval was performed with citrate buffer (pH 6.0). Anti-acetyl histone H3 (Lys9 and Lys27) antibody was diluted 1/1000 with SignalStain Antibody Diluent (#8112; Cell Signaling Technology) and incubated for 1 h at room temperature. Antigen–antibody reactions were visualized with SignalStain Boost IHC Detection Reagents (HRP, Rabbit #8114; Cell Signaling Technology).
Heat-induced epitope retrieval was performed with citrate buffer (pH 6.0). Anti-acetyl histone H3 (Lys9 and Lys27) antibody was diluted 1/1000 with SignalStain Antibody Diluent (#8112; Cell Signaling Technology) and incubated for 1 h at room temperature. Antigen–antibody reactions were visualized with SignalStain Boost IHC Detection Reagents (HRP, Rabbit #8114; Cell Signaling Technology).
6
Immunohistochemistry and Immunofluorescence Protocols
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For immunohistochemistry (IHC), sections were deparaffinized and rehydrated before undergoing heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 30 min. Slides were blocked for endogenous peroxidase for 20 min, then blocked for 1 h in 5% BSA, 1% goat serum, 0.1% Tween-20 buffer in PBS, and incubated overnight at 4 °C in primary antibody diluted in blocking buffer or SignalStain® Antibody Diluent (Cell Signaling). Slides were incubated in biotinylated secondary antibodies for 1 h at room temperature and signal was detected using the Vectastain Elite ABC kit (Vector Laboratories). Ki67 (Cell Signaling Cat#9027 S) was used at dilution 1:500. For immunofluorescence (IF), cells or tissue sections were fixed by 4% paraformaldehyde for 5 min, blocked for 1 h and incubated overnight at 4 °C in primary antibody diluted in blocking buffer. Slides were then incubated for 1 h at room temperature in Alexa Fluor-488 secondary antibody (Invitrogen R37118) at 1:500 dilution in blocking buffer and mounted using mounting media with DAPI (EMS).
7
STAT1 Immunohistochemistry Protocol
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After deparaffinization and rehydration, 3 to 5 μm thick tissue sections were placed for 10 min in 10 mM citrate buffer (pH 6.0) at 95°C for antigen unmasking, followed by treatment with 3% H2O2 for 10 min at room temperature to block endogenous peroxidase. Staining for STAT1 was performed with the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) using a 1:250 dilution of primary STAT1 rabbit monoclonal antibody (42H3, Cell Signaling) in SignalStain® Antibody Diluent (Cell Signaling) and overnight incubation at 4°C. Slides were treated with the chromogen diaminobenzidine and counterstained with Mayer’s Hemalum (Merck).
8
Immunohistochemical Analysis of pERK and pS6 in Xenografts
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We carried out immunohistochemistry on paraffin‐embedded blocks of xenografted tumor tissues using rabbit polyclonal antibodies against pERK diluted 1:100 (Cell Signaling Technology) and pS6 diluted 1:400 (Cell Signaling Technology), as described previously (ref: GC CGH), with slight modifications. Briefly, after antigen retrieval and inactivation of endogenous peroxidase, tissue sections were blocked with 2.5% horse serum (ImmPRESS reagent kit; Vector Laboratories, Burlingame, CA, USA) at room temperature for 30 min and then incubated at 4°C overnight with antibodies against pERK and pS6 diluted in SignalStain Antibody Diluent (Cell Signaling Technology) and 1% BSA/PBS, respectively. After washing in PBS, the sections were incubated at room temperature for 30 min with peroxidase‐conjugated horse anti‐rabbit Ig (ImmPRESS reagent kit; Vector Laboratories). Peroxidase activity was detected using ImmPACT DAB Peroxidase Substrate (Vector Laboratories).
9
Immunohistochemistry of Paraffin-Embedded Tumor Sections
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IHC examination of paraffin-embedded tumor sections was performed by deparaffinizing the sections with washes of xylene, and rehydrating them with serial washes of absolute ethanol, 95% ethanol, and then with distilled water. Antigen retrieval was performed by boiling the sections in Tris-EDTA (pH 9) for ST2 staining and in acetate (pH 6) for other antibodies. Endogenous peroxidase was blocked with 3% oxygen peroxidase. Non-specific binding was blocked with Background Buster (Innovex Biosciences, Richmond, CA). The primary antibody was incubated on slide overnight at 4°C in SignalStain antibody diluent (Cell Signaling Technology). Secondary antibody binding and 3,3′-diaminobenzidine (DAB) development were performed the following day. The nuclei were stained with hematoxylin. The slides were dehydrated, mounted and visualized under an Olympus BX41 microscope (Olympus) with a CCD camera. Primary antibodies included those used in the IF experiments, as well as an antibody against the full-length ST2 (prepared for the present study by Genemed Synthesis Inc.). Secondary antibodies included anti-rabbit horseradish peroxidase (HRP; Cell Signaling Technology) diluted in SignalStain® Boost IHC Detection Reagent (Cell Signaling Technology) with DAB as the substrate, and Eukitt® as the mounting media (both from Sigma-Aldrich, St. Louis, MO, USA).
10
Immunohistochemistry Protocol for H3K27me3 Staining
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