Blocks were sectioned at a thickness of 3 μm. Antibodies targeting CD68 (clone PG-M1, Dako), phosphorylated signal transducer and activator of transcription 1 (phospho-STAT1; monoclonal, clone, and 58D6, Cell Signaling Technology, Danvers, MA, USA), and c-Maf (clone EPR16484, Abcam, Cambridge, UK) were used for analyses. Double staining was performed using a Dako Envision+ system with dextran polymers conjugated with horseradish peroxidase (Dako), as previously described.12 (link) First, sections were stained with anti-CD68 antibodies for 30 min at room temperature, generating a brown color. Denaturing solution (BioCare Medical-CA, USA) was added for 5 min at room temperature for elution during double staining. Antigen retrieval was performed by heat treatment for 45 min with HIER T-EDTA Buffer (Dako). After incubation, sections were reacted with phospho-STAT1- or c-Maf-specific reagents using dextran polymers conjugated with horseradish peroxidase (Dako) overnight at 4 °C, using a Vina Green Chromogen Kit (BioCare Medical-CA), which produced green staining. Finally, slides were washed in Wash Buffer (Dako) for 3 min. Sections were counterstained with hematoxylin. The antibodies used in this study are listed in Supplementary Table 1.
Horseradish peroxidase
Manufactured by Agilent Technologies
Sourced in Denmark, United States, Germany, Japan, United Kingdom
Horseradish peroxidase is an enzyme commonly used as a label or reporter in various biochemical and immunological assays. It catalyzes the oxidation of substrates, such as chromogenic or chemiluminescent compounds, in the presence of hydrogen peroxide.
Automatically generated - may contain errors
Lab products found in correlation
Envision system, by Agilent Technologies (3 mentions)
Nupage, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Cytiva (3 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (3 mentions)
Whatman, by Cytiva (3 mentions)
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (2 mentions)
Ecl plus detection reagent, by Santa Cruz Biotechnology (2 mentions)
Nupage bis tris, by Thermo Fisher Scientific (2 mentions)
Lsab kit, by Agilent Technologies (2 mentions)
β actin, by Merck Group (2 mentions)
Protran, by Cytiva (2 mentions)
Pannoramic 250 flash 2, by 3DHISTECH (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Microm hm 340e, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ccp58, by Leica (2 mentions)
Nbp1 90156, by Novus Biologicals (2 mentions)
Microwave processor, by Bio-Rad (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
94 protocols using horseradish peroxidase
1
Multicolor Immunohistochemistry Analysis
2
Quantifying Autophagy Markers in Lung and Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For lung cancer patients’ tissue, the sections (5 µm) from the tissue microarray blocks were labelled with antibodies. The LC3B antibody was diluted 1:2000. An HRP- conjugated secondary antibody (Beijing Sequoia Jinqiao Biological Technology Co., Ltd.) was used. The specific target was visualized with a 3, 3’-diaminobenzidine (DAB) detection kit (Beijing Sequoia Jinqiao Biological Technology Co., Ltd.) and counterstained with hematoxylin. Microphotographs from each arrayed tissue were taken with a fixed exposure time and color balance to ensure consistency. LC3B production was quantified using ImagePro Plus9.1 (Media Cybernetic, Silver Spring, MD).
For animal tumor tissue, paraffin sections were first stained with anti-ATG5 (#ab108327, 1:50; Abcam, Cambridge, MA, USA) and anti- LC3B (#ab48394, 1:50; Abcam). Horseradish peroxidase (DAKO, Glostrup, Denmark) was then used as a secondary antibody. When an antigen-antibody-antibody complex was formed, a substrate of the peroxidase, diaminobenzidine, was added as chromogen. The staining was performed according to the manufacturer’s instructions. The pictures were taken at a magnification of 400 × and analyzed using Image Pro-plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). Five different pictures were measured for each sample.
For animal tumor tissue, paraffin sections were first stained with anti-ATG5 (#ab108327, 1:50; Abcam, Cambridge, MA, USA) and anti- LC3B (#ab48394, 1:50; Abcam). Horseradish peroxidase (DAKO, Glostrup, Denmark) was then used as a secondary antibody. When an antigen-antibody-antibody complex was formed, a substrate of the peroxidase, diaminobenzidine, was added as chromogen. The staining was performed according to the manufacturer’s instructions. The pictures were taken at a magnification of 400 × and analyzed using Image Pro-plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). Five different pictures were measured for each sample.
3
Quantification of Oxidized Proteins by OxyBlot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oxidized proteins were detected by OxyBlot assay using the OxyBlot protein oxidation detection kit (Merck Millipore, Darmstadt, Germany) as previously described [55 ,56 ]. Briefly, proteins (with an equal amount) derived from each sample were derivatized with 2,4-dinitrophenylhydrazine (DNPH), resolved by 12% SDS-PAGE, and transferred onto a nitrocellulose membrane. Non-specific bindings were blocked with 5% skim milk/PBS for 1 h at 25°C. The membrane was then incubated with a rabbit polyclonal anti-dinitrophenyl (DNP) antibody (diluted 1:500 in 1% BSA/PBS) at 4°C overnight, followed by a corresponding secondary antibody conjugated with horseradish peroxidase (Dako) (diluted 1:1,000 in 1% BSA/PBS) at 25°C for 1 h. After washing with PBS three times, the immunoreactive protein bands were developed by SuperSignal West Pico chemiluminescence substrate (Pierce Biotechnology, Rockford, IL) and visualized by autoradiogram.
4
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes and immunoblotted overnight at 4 °C with the primary antibodies (1:500), rinsed with TBST, and incubated with 1:5000 secondary antibodies conjugated to horseradish peroxidase (Dako). After applying electrochemiluminescence (ECL) Plus detection reagents (Santa Cruz Biotechnology), protein bands were visualized using X-ray film (Fujifilm, Tokyo, Japan). GAPDH-specific monoclonal antibody (1:2000; Santa Cruz Biotechnology) was used as the internal control.
5
Western Blotting Protocol for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blotting, cells were washed with cold PBS, lysed in NP-40 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) supplemented with protease inhibitors and centrifuged (20,000 g for 15 min). Protein concentration was measured using a BCA protein assay kit (Pierce) and LDS sample buffer (NuPAGE, Invitrogen) was added to the lysate or directly to the medium. Equal amounts were loaded on SDS-PAGE pre-cast gradient gels (4–12% Nu-Page Bis-Tris, Invitrogen), followed by transfer to nitrocellulose membrane. Non-specific protein binding was blocked by 5% skimmed milk in TBST followed by incubation with primary antibodies were overnight at 4°C in TBST with 2.5% skimmed milk, and then secondary antibodies conjugated to horseradish peroxidase (DAKO, Glostrup, Denmark) for 1 h at room temperature. Proteins were detected using ECL western blot reagent.
6
HIV Protein Immunoblot Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study are a mouse anti-CAp24 and a rabbit anti-MAp17 [NIH, USA], the rabbit anti-IN was a gift of Dr JF.Mouscadet (LBPA, ENS Cachan), the rabbit anti-RT was a gift of Dr JL.Darlix (ENS Lyon). The anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase [Dako ] were used for immunoblot detection.
7
Immunohistochemical Analysis of ANXA2 and HO-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TMA was processed and fixed using routinely established protocols and stained as previously described [28 (link)]. Briefly, immunohistochemistry was done using the streptavidin-biotin-peroxidase complex system LSAB + kit, horseradish peroxidase (DAKO). Endogenous peroxide activity was quenched using hydrogen peroxide in distilled water (3%). Antigen retrieval was done by microwaving. Tissue slides were incubated overnight with the following primary antibodies: monoclonal mouse anti-ANXA2 (1:200) from Cell Signaling Tech, (Danvers, MA, USA) and rabbit polyclonal anti–HO-1 (1:50) from Abcam (Burlingame, CA, USA); this was followed by sequential incubations with biotinylated link antibody and peroxidase-labeled streptavidin complex. The peroxidase reaction was conducted, under microscope, using 3,3′-diaminobenzidine. Slides were counter-stained with Mayer’s hematoxylin and analyzed by standard light microscopy. Negative control slides were prepared by substituting primary antiserum with PBS. For semiquantitative analysis, the degree of staining was rated as high, moderate, low, or not detectable (3+, 2+, 1+, and 0, respectively); the staining was also observed for localization.
8
Immunohistochemistry for GR and HO-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical techniques were performed as previously described [6 (link),11 (link)]. Briefly, immunohistochemistry was done using the streptavidin-biotin-peroxidase complex system LSAB + kit, horseradish peroxidase (DAKO, Santa Clara, CA, USA). Endogenous peroxide activity was quenched using hydrogen peroxide in distilled water (3%). Antigen retrieval was done by microwaving. Tissue slides were incubated overnight with GR and HO-1 primary antibodies. This was followed by sequential incubations with biotinylated antibody and peroxidase-labelled streptavidin complex. The peroxidase reaction was conducted, under microscope, using 3,3′-diaminobenzidine. Slides were counterstained with Mayer’s hematoxylin and analyzed by standard light microscopy. Negative control slides were prepared by substituting primary antiserum with PBS. For semiquantitative analysis, the degree of staining was scored as high, moderate, low, or not detectable (3+, 2+, 1+, and 0, respectively); staining localization was also recorded. We considered positive expression when more than 10% cells exhibited positive staining.
9
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates and homogenized tissue lysates were prepared in ice-cold Lysis-M buffer (Roche, Mannheim, Germany), supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche). The whole process after protein separation was performed as previously described [56 (link)]. We used 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for protein separation. The primary antibodies and the dilutions used were: CTGF (Santa Cruz Biotechnology, Santa Cruz, CA; 14939, 1:200), X-linked inhibitor of apoptosis (Abcam, Cambridge, UK; 21278, 1:1,000), β-actin (Sigma-Aldrich, St. Louis; A1978, 1:1,000). Secondary antibodies were immunoglobulins conjugated to horseradish peroxidase (Dako, Glostrup, Denmark; 1:1,000). Immunodetection was performed using Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Densitometric analysis was performed using the ImageJ software (http://rsb.info.nih.gov/ij/ ).
10
Double Immunohistochemistry for α-SMA and Osteopontin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hydration was followed by antigen retrieval (Target Retrieval Solution, DAKO) and block of endogenous peroxidases. Application of goat serum (45’, 1/5, DAKO) blocks nonspecific sites. The first primary antibody was applied overnight (1/500, rabbit anti-α-SMA, Abcam ab5694) followed by incubation with Goat anti Rabbit IgG, conjugated with Horseradish peroxidase (45’, 1/100, DAKO P0448) containing 10% mouse serum (Sigma) and visualization by 3,3’-Diaminobenzidine (DAKO K346711). After thorough rinsing and block with donkey serum (45’, 1/5, Sigma), the second primary antibody is applied overnight (1/200, Goat anti-osteopontin, R&D AF808). The next day, incubation of the biotinylated secondary antibody (45’, 1/300, DAG-B, Santa Cruz SC2042) is followed by signal amplification (30’, Streptavidin-Alkaline Phosphatase, Abcam 64268) and visualization using green chromogen (Enzo ADI-950-160-1). Slides are mounted with Faramount (DAKO, S3025). Every section was divided in four quadrants, each of which was scored (1: no osteopontin, (Fig 2A ) to 5: maximal osteopontin (Fig 2B )) for osteopontin presence. Quadrant scores were averaged in a blinded manner and used for analysis of SMC phenotype. Negative control stainings (no primary antibody, data not shown) were thoroughly studied before, to avoid taking blood remains into account in our scoring system.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!