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).
Blocking solution
Manufactured by Cell Signaling Technology
Sourced in United States
Blocking solution is a laboratory reagent used to prevent non-specific binding of antibodies or other proteins in immunoassays and western blotting. It contains a mixture of proteins or other compounds that help to block unoccupied binding sites on the membrane or support material, reducing background signal and improving specificity of the target analyte detection.
Automatically generated - may contain errors
Lab products found in correlation
Signalstain boost detection reagent, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Signalstain dab, by Cell Signaling Technology (1 mentions)
Vectamount aq, by Vector Laboratories (1 mentions)
Immunohistochemistry protocol paraffin, by Cell Signaling Technology (1 mentions)
Alexa 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mouse anti pakt, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Triton 100, by Merck Group (1 mentions)
Signalstain dab reagent, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
Nanog, by Cell Signaling Technology (1 mentions)
Iblot gel transfer system, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Novex tris glycine sds sample buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using blocking solution
1
Immunohistochemical Analysis of Pgp and CD44 in Ovarian Cancer
2
Immunofluorescent Protein Detection
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Tissue sections were prepared as described above followed by incubation (3 h at 37 °C and ON at 4 °C) with mouse anti-p-Akt (cat # 4051) and rabbit polyclonal anti-caspase-3 primary antibodies, 1:150 in blocking solution (Cell Signaling Technology, cat # 9662, MA, USA). After washing, the slides were incubated (37 °C for 30 min) with Cy3 conjugated goat anti-mouse (for p-Akt) and Alexa 488 conjugated goat anti-rabbit (Invitrogen, cat # A-11008, Carlsbad, CA, USA) secondary antibodies for caspase-3. The procedure was then followed as described above.
3
Immunohistochemical Staining of Paraffin Sections
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Paraffin embedded specimens were deparaffinized in xylene, subjected to heat mediated antigen-retrieval in 10 mM sodium citrate (pH 6.0), permeabilized in 0.2% Triton-100 (Sigma) and treated with 100–400 μl blocking solution (Cell Signal Technology). After removal of blocking solution, tissue sections were incubated with 100–400 μl primary antibody as indicated in Figure 5 overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibody (Cell Signal Technology). The signal was amplified with SignalStain Boost Detection Reagent (Cell Signal Technology) and detected using SignalStain DAB reagent (Cell Signal Technology).
4
Western Blot Analysis of Stem Cell Markers
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Cells were lysed in the RIPA lysis Buffer (Thermo Scientific) supplemented with 1X Phosphatase Inhibitor (Thermo Scientific) and Protease Inhibitor (Thermo Scientific). The protein lysates were collected following the centrifugation.
The proteins were mixed with Novex Tris-Glycine SDS sample buffer (Invitrogen) and separated on the 8% Tris-glycine gels (Invitrogen). The iBlot Gel Transfer system (Invitrogen) was used to transfer protein onto the nitrocellulose membrane (Invitrogen). Subsequently, the membranes were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS)/0.1% Tween-20 (Cell signaling Technology) for one hour. Primary antibodies were then diluted in the blocking solution and applied overnight at 4 oC. The antibodies used in this study were: GAPDH (the loading control; 1:1000; Millipore); Oct3/4 (1:200; Santa Cruz); Nanog (1:1000; Cell signaling Technology); Rb (1:1000; Cell signaling Technology); SV-40 T121 (1:1000; Abcam); and GFP (1:200; Santa Cruz). After the membranes were washed in TBS/0.1% Tween-20 at room temperature on the second day, HRP-conjugated secondary antibodies were diluted in blocking solution (1:1000, Cell Signaling) and added to the membranes for one hour. ECL-Prime Western Blotting Detection Reagent (GE Healthcare) was used for signal detection.
The proteins were mixed with Novex Tris-Glycine SDS sample buffer (Invitrogen) and separated on the 8% Tris-glycine gels (Invitrogen). The iBlot Gel Transfer system (Invitrogen) was used to transfer protein onto the nitrocellulose membrane (Invitrogen). Subsequently, the membranes were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS)/0.1% Tween-20 (Cell signaling Technology) for one hour. Primary antibodies were then diluted in the blocking solution and applied overnight at 4 oC. The antibodies used in this study were: GAPDH (the loading control; 1:1000; Millipore); Oct3/4 (1:200; Santa Cruz); Nanog (1:1000; Cell signaling Technology); Rb (1:1000; Cell signaling Technology); SV-40 T121 (1:1000; Abcam); and GFP (1:200; Santa Cruz). After the membranes were washed in TBS/0.1% Tween-20 at room temperature on the second day, HRP-conjugated secondary antibodies were diluted in blocking solution (1:1000, Cell Signaling) and added to the membranes for one hour. ECL-Prime Western Blotting Detection Reagent (GE Healthcare) was used for signal detection.
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