Immunohistochemistry was done as described previously [9] . LV tissues were fixed in 10% neutral buffered formalin. The tissues were dehydrated and embedded in paraffin and thin tissue sections (4 μm) were made. After the deparaffinized sections were heated using a microwave for antigen retrieval, endogenous peroxidase activity was blocked by incubating in Dako REAL peroxidase-blocking solution (Dako, Glostrup, Denmark) for 15 min. Then, the sections were blocked with 5% normal goat serum for 60 min and subsequently incubated with specific primary antibody against total-eEF2K (1:250 dilution), phospho-eEF2K (1:250 dilution) or phospho-eEF2 (1:200 dilution) at 4 °C overnight. After washed in Tris buffer, the sections were incubated in biotinylated link (Dako) for 10 min and next in streptavidin-HRP (Dako) for 10 min at room temperature. The images were visualized by a liquid DAB + substrate chromogen system (Dako) and obtained using a CCD-camera equipped light microscope (BX-51). For comparing expression and phosphorylation of eEF2K and eEF2 phosphorylation between LV from ISO and Cont group, ratio of total-eEF2K-, phosphorylated eEF2K- or phosphorylated eEF2-positive area in three fields from each LV section was calculated using Image J software (NIH, Bethesda, MD, USA).
Dako real peroxidase blocking solution
Manufactured by Agilent Technologies
Sourced in United States
Dako REAL peroxidase-blocking solution is a laboratory product designed to inhibit endogenous peroxidase activity in tissue samples. It is intended for use in immunohistochemical and in situ hybridization procedures.
Automatically generated - may contain errors
Lab products found in correlation
Target retrieval solution, by Agilent Technologies (5 mentions)
Ab56299, by Abcam (2 mentions)
Dako protein blocking solution, by Agilent Technologies (2 mentions)
Envision kit, by Agilent Technologies (2 mentions)
Anti foxp3 clone pch101, by Thermo Fisher Scientific (2 mentions)
Kaiser s glycerol gelatin, by Carl Roth (2 mentions)
Streptavidin hrp, by Agilent Technologies (1 mentions)
Biotinylated link, by Agilent Technologies (1 mentions)
Liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Dako protein block, by Agilent Technologies (1 mentions)
Lsab2 system hrp, by Agilent Technologies (1 mentions)
3 39 diaminobenzidine, by Agilent Technologies (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Dako real antibody diluent, by Agilent Technologies (1 mentions)
Pab12714, by Abnova (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Aec high sensitivity substrate chromogen, by Agilent Technologies (1 mentions)
Sc 1178, by Santa Cruz Biotechnology (1 mentions)
Mmp13, by Proteintech (1 mentions)
24 protocols using dako real peroxidase blocking solution
1
Immunohistochemical Analysis of eEF2K and eEF2 in LV Tissue
2
Tumor Tissue Immunohistochemistry Protocol
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Ki67 and CD-31 IHC of tumor sections was performed as previously described [14 (link)]. Paraffin sections (4-μm-thick) of the biggest tumor sections from ID8, ID8-c-MYC, and ID8-KRAS mice (sections were taken when BW exceeded 23 g) were dewaxed in xylene and rehydrated through graded ethanol to water. Antigens were retrieved by boiling in 10 mM citrate buffer (pH 6.0) for 30 min. The cooled sections were incubated in DAKO REAL Peroxidase-Blocking solution (DAKO, Carpinteria, CA, USA) for 10 min to quench endogenous peroxidase. Sections were incubated in DAKO Protein Blocking solution (DAKO) at room temperature for 10 min to block non-specific binding. Sections were then stained for Ki67 using rabbit monoclonal antibody against mouse Ki67(1:100; Spring Bioscience, CA, USA), CD-31 using a rat monoclonal antibody against mouse CD-31 (ab56299, Abcam, Tokyo, Japan, 1:100 dilution).
3
Immunohistochemical Analysis of Osteopontin and Osteoprotegerin
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The methodology is analogous to that described in Kuzan et al. 2021 and Kuzan et al. 2017 [8 (link),9 (link)]. Briefly, slides were dewaxed with xylene, hydrated with ethanol solutions, the antigens were discovered with Proteinase K (Dako, Minneapolis, MN, USA), endogenous peroxidase was blocked with Dako Real Peroxidase-Blocking solution and non-specific sites with DakoProtein Block (Dako). Then, reactions with the primary antibodies were carried out using Monoclonal Anti-human Osteopontin (R&D Systems, MAB14332) and Monoclonal Anti-human Osteoprotegerin/TNFRSF11B Antibody (R&D Systems, MAB8051). Then, the LSAB2 System-HRP, DakoCytomation kit was used for the next steps of immunohistochemical reactions, followed by an enzymatic reaction with the DAB substrate (3,39-diaminobenzidine, Dako North America, Carpinteria, CA, USA). The basophilic structures by Delafield’s hamatoxylin (Hematoxylin solution according to Delafield, Sigma) were stained, rinsed in tap water, dehydrated and fixed with ethanol and xylene, and sealed in DPX.
4
Immunohistochemical Analysis of Tumor Angiogenesis
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Paraffin sections (4 µm) of TC-1 tumors were dewaxed in xylene and rehydrated through graded ethanol to water. Antigens were retrieved by boiling in 10 mM citrate buffer (pH 6.0) for 30 min. The cooled sections were incubated in DAKO REAL Peroxidase-Blocking solution (DAKO, Carpinteria, CA, USA) for 10 min to quench endogenous peroxidase. To block nonspecific binding, sections were incubated in DAKO Protein Blocking solution (DAKO) for 10 min at room temperature. Sections were then incubated with a rabbit polyclonal antibody against mouse MMP-9 (PAB12714, Abnova, 1∶100 dilution) in DAKO REAL Antibody Diluent (DAKO) overnight at 4°C. The slides were incubated for 1 hour at room temperature with peroxidase-conjugated secondary antibodies, washed, incubated with DAB, counterstained with hematoxylin, dehydrated through an ethanol series and xylene, and mounted. To evaluate tumor microvessel formation, tumor sections were stained for CD-31 using a rat monoclonal antibody against mouse CD-31 (ab56299, Abcam, Tokyo, Japan, 1∶100 dilution).
5
Histological Assessment of Capsular Tissue
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Hematoxylin-eosin staining and immunohistochemical staining were performed for
the histological assessment of capsular tissue. After overnight fixing with 10%
buffered formalin, the sample was washed and dehydrated through a graded series
of alcohol. The specimen was embedded in paraffin and cut into 4-µm thickness
for the attachment to the slide. It was treated with Dako REAL
Peroxidase-Blocking Solution (DAKO) for 15 minutes, 0.1% Triton x-100 solution
for 10 minutes, and 10% normal goat serum (Vector Laboratories) for 20 minutes.
The primary antibody mouse monoclonal antihuman type I and III collagen,
fibronectin, and MMP-2 and MMP-9 were incubated overnight at 4°C. After washing
with phosphate-buffered saline, biotinylated secondary antibody (Vectastain
Elite ABC Kit; Vector Laboratories) was applied for 20 minutes. To catalyze
chromogen development in 3,3′-diaminobenzidine tetrachloride, streptavidin
conjugated peroxidase was applied. The stained sections were examined, and the
distribution of type I and III collagen, fibronectin, MMP-2, and MMP-9 was
determined under optical microscopy after counterstaining with
hematoxylin-eosin.
the histological assessment of capsular tissue. After overnight fixing with 10%
buffered formalin, the sample was washed and dehydrated through a graded series
of alcohol. The specimen was embedded in paraffin and cut into 4-µm thickness
for the attachment to the slide. It was treated with Dako REAL
Peroxidase-Blocking Solution (DAKO) for 15 minutes, 0.1% Triton x-100 solution
for 10 minutes, and 10% normal goat serum (Vector Laboratories) for 20 minutes.
The primary antibody mouse monoclonal antihuman type I and III collagen,
fibronectin, and MMP-2 and MMP-9 were incubated overnight at 4°C. After washing
with phosphate-buffered saline, biotinylated secondary antibody (Vectastain
Elite ABC Kit; Vector Laboratories) was applied for 20 minutes. To catalyze
chromogen development in 3,3′-diaminobenzidine tetrachloride, streptavidin
conjugated peroxidase was applied. The stained sections were examined, and the
distribution of type I and III collagen, fibronectin, MMP-2, and MMP-9 was
determined under optical microscopy after counterstaining with
hematoxylin-eosin.
6
Immunohistochemical Analysis of Progenitor Cell Markers
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Immunohistochemistry was performed as previously described [27 (link)]. Heat-induced epitope retrieval was performed in a retrieval buffer (Dako Target Retrieval 10×, S1699, Dako, USA), and endogenous peroxidase was blocked using a peroxidase-blocking solution (Dako REAL Peroxidase-Blocking Solution, S2023, Dako, USA). Next, the PRF sections were incubated overnight with primary antibodies against fibrinogen (1:100, Santa Cruz, sc-69775), CD45, and Oct-4 (1:100, Santa Cruz, sc-1178), and then incubated for 45 min with biotinylated secondary antibodies (1:1000). Visualization was achieved using 3-amino-9-ethyl, a high-sensitive substrate chromogen (AEC+ High-Sensitivity Substrate Chromogen, ready-to-use, K346911-2, DAKO, Glostrup, Denmark).
7
Paraffin Sectioning and Antigen Retrieval
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For thin paraffin sections, mouse brains were paraffine embedded and cut into 2 um thin sections. After deparaffination, antigen retrieval was performed using a Pascal Citrate buffer at pH6.0. To inactivate endogenous peroxidases, slides were incubated with Dako REAL peroxidase-blocking solution (Dako, K0672) and subsequently blocked with 10% FCS in PBS.
8
Immunohistochemical Analysis of Oxidative Stress Markers
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Antigen retrieval was performed on deparaffinized sections using the Target Retrieval Solution (Dako, USA). After quenching in Dako REAL™ Peroxidase Blocking Solution (Dako, USA), the slides were incubated with primary antibodies against Nrf2 (Affinity, China), HO-1 (Affinity, China), MMP-3 (Proteintech, China), and MMP-13 (Proteintech, China) overnight at 4°C. Following three PBS washes, the sections were incubated with the corresponding secondary antibody for 50 min followed by an examination on the liquid diaminobenzidine (DAB) + substrate-chromogen system. Leica photomicroscope (Leica, Germany) was used to capture the images.
9
Immunohistochemistry of Lung Carcinoma
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IHC was performed on formalin-fixed, paraffin-embedded tissue sections of 16 lung carcinomas and 3 normal lung controls from the Pathology Service of the Hospital Clinic of Barcelona after review by a thoracic pathologist. 4-μm-thick transverse sections of formalin-fixed, paraffin-embedded tissue were serially cut and mounted onto Dako Silanized Slides (S3003; Dako, Glostrup, Denmark). For antigen retrieval, the sections were manually immersed in Target Retrieval solution, high pH (Dako) and heated in a water bath at 95–99uC for 20 min. Endogenous peroxidase activity was quenched by immersion in Dako Real Peroxidase-Blocking solution for 10 min. The tissue sections were incubated with primary antibody against TTF1 (1:100, 8g7g3/1, DAKO, glostrop, Denmark). The slides were then washed in Tris–HCl and detected with horseradish peroxidase-conjugated anti-rabbit EnVision + kit (DAKO). Finally, sections were stained with hematoxylin. All slides were blindly scored by the same two pathologists. Nuclear staining of tumor cells was considered TTF1+. Tumors with completely no TTF1 expression in nuclei were de ned as TTF1 − .
10
Immunohistochemical Analysis of Bone Remodeling Factors
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Sections were deparaffinized in xylene and rehydrated in gradient alcohol, and underwent heat-induced antigen retrieval using Target Retrieval Solution (Dako, Carpinteria, CA). The slides were then quenched in Dako REAL™ Peroxidase-Blocking Solution (Dako) and blocked in 5% goat serum (Invitrogen, USA) followed by incubation with primary antibodies anti-RANKL (Acan, 1:50, service bio, Inc., Wuhan, China), anti-BMP2 (1:100, service bio, Inc., Wuhan, China) and anti-transforming growth factor beta (TGF-β)-1 (1:100, service bio, Inc., Wuhan, China) at 4°C overnight. Subsequently, the sections were incubated with horseradish peroxidase conjugated IgG H&L secondary antibody (1:1000, Abcam) for 1 h in room temperature and visualized using the liquid 3, 30-diaminobenzidine (DAB)+ substrate-chromogen system (Dako).
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