Peroxidase block solution

Manufactured by Agilent Technologies

Sourced in Germany

Peroxidase block solution is a laboratory reagent used to inactivate endogenous peroxidase activity in biological samples prior to immunohistochemical or immunocytochemical staining procedures. It helps to prevent non-specific binding of antibodies and reduce background staining.

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Peroxidase block (48 citations) Block solution (4 citations)

4 protocols using peroxidase block solution

Immunohistochemical Analysis of CD3 and Orai-1 in Skin Tissue

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Skin tissue sections were deparaffinized in xylene and dehydrated in graded alcohol. After washing the samples in PBS, sections were placed in epitope-retrieval buffer (DakoCytomation, Carpenteria, CA, USA) at 95°C for 20 min and subsequently cooled to room temperature (RT) for an additional 20 min. The sections were blocked with 10% goat serum in PBS, followed by blocking for endogenous peroxidases stained with peroxidase block solution (DakoCytomation). Sections were incubated overnight at 4°C with antibodies against anti-CD3 and anti-Orai-1 (Cell Signaling Co., Danvers, USA). Unbound antibodies were removed the following day by washing the slides three times with PBS. Areas positive for CD3 and Orai-1 induction were stained brown after development with diaminobenzidine. The slides were counterstained with filtered Mayer's hematoxylin (Sigma-Aldrich, St. Louis, MO), rinsed with distilled water, allowed to dry, and then mounted for viewing purposes. The images were observed using a Leica digital camera and microscope (Leica Co., Wetzlar, Germany).

Kang S.Y., Jung H.W., Nam J.H., Kim W.K., Kang J.S., Kim Y.H., Cho C.W., Cho C.W., Park Y.K, & Bae H.S. (2017). Effects of the Fruit Extract of Tribulus terrestris on Skin Inflammation in Mice with Oxazolone-Induced Atopic Dermatitis through Regulation of Calcium Channels, Orai-1 and TRPV3, and Mast Cell Activation. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 8312946.

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Immunohistochemical Analysis of Endometrial Biopsies

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Paraffin-embedded endometrial biopsies from the control and uRPL groups were sectioned at 4 μm in a microtome and deposited on SuperFrost slides (Menzel, Germany). The investigated markers were assessed in subsequent sections. Following dewaxing in xylene and rehydration through descending ethanol concentrations, antigen retrieval was achieved in a citrate buffer at >95 °C for 15 min. Slides were washed in Tris-buffered saline-Tween20 0.05% (TBST). For inhibition of endogenous peroxidase activity, and tissue sections were incubated with peroxidase block solution (Dako, Germany) for 10 min and washed in TBST. Primary antibodies were prepared in antibody diluent solution (Dako, Germany) and incubated for 1 h at room temperature (RT). Antibody specifications and dilutions are shown in Table 1.
Following washing in TBST, the slides were incubated with the secondary antibody (labelled polymer-horseradish peroxidase (HRP) anti-mouse, clone: DAK-GO1, Dako, Germany) for 30 min at RT. The peroxidase reaction was developed with DAB (3,3′-diaminobenzidine; Dako, Germany) and discontinued with water after 15 min. Counterstaining with hematoxylin was followed by mounting of slides with histofluid and cover slip.

Chiokadze M., Bär C., Pastuschek J., Dons’koi B.V., Khazhylenko K.G., Schleußner E., Markert U.R, & Favaro R.R. (2020). Beyond Uterine Natural Killer Cell Numbers in Unexplained Recurrent Pregnancy Loss: Combined Analysis of CD45, CD56, CD16, CD57, and CD138. Diagnostics, 10(9), 650.

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Immunocytochemical Analysis of PHGDH

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For immunocytochemical analysis, cultured cells grown in a 12-well plate were fixed with 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 30 min, washed in PBS and then treated with peroxidase block solution (DAKO, Carpinteria, CA) to inactivate endogenous peroxidase activity. Following a PBS wash, cells were permeabilized with 0.1% Triton-X-100 (Fisher Scientific, Pittsburgh, PA) and non-specific proteins were blocked in 2% BSA for 15 min at room temperature (RT). This step was followed by incubation in a humidified atmosphere at 37 °C for 1 hour with primary monoclonal mouse antibody to PHGDH (Santa Cruz Biotechnology) diluted 1:15 in PBS. After washing the cells with PBS, the immunocytochemical analysis for PHGDH was conducted for 30 min at RT using DAKO EnVision + System-HRP. After washing the cells with PBS again, the immunoreactions were visualized with 3,3′-diaminobenzidine (DAB) solution (DAKO). Images were captured with the 40X objective lens on the TS100 microscope (Nikon, Tokyo, Japan). Negative controls, in which the primary antibodies were replaced with PBS and non-immune sera, did not show nonspecific staining.

Singh M., Warita K., Warita T., Faeder J.R., Lee R.E., Sant S, & Oltvai Z.N. (2018). Shift from stochastic to spatially-ordered expression of serine-glycine synthesis enzymes in 3D microtumors. Scientific Reports, 8, 9388.

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Immunohistochemical Analysis of Matrigel Nodules

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The matrigel nodules harvested from mice were fixed in 10% neutral-buffered formalin for 16–18 h. The fixed tissue samples were transferred to 70% ethanol and dehydrated in 100% ethanol. The dehydrated tissue samples were cleared in xylene, infiltrated, and embedded in paraffin. Serial sections were cut at 3–5 µm for use in immunohistochemical protocols. Prior to immunostaining, sections were immersed in preheated Target Retrieval Solution (Dako, Carpinteria, CA) and heated in a steamer for 20 min. The sections were allowed to cool to room temperature and immersed into Tris buffered saline (TBS) plus 5% Tween 20 (TBS-T) for 5 min. Blocking was performed by incubating the sections in Dako Peroxidase block solution. The slides were washed and incubated with the primary antibody overnight at 4 °C. The list of the primary antibodies used, along with their catalogue numbers and dilution is listed in Supplemental Materials Table 2 (S2 Table). After washing, the slides were incubated with DakoCytomation EnVision+ dual Link Antibody or Dako HRP-labelled antibody at room temperature for 30 min. Liquid diaminobenzidine (Dako) was used for visualization. Counter staining was performed for 8 min at room temperature using Ready-to-use Hematoxylin (Dako). Slides were rinsed with distilled water, dehydrated in graded ethanol, cleared in xylene, and cover-slipped.

Shrestha S., Garrett S.H., Sens D.A., Zhou X.D., Guyer R, & Somji S. (2019). Characterization and determination of cadmium resistance of CD133+/CD24+ and CD133−/CD24+ cells isolated from the immortalized human proximal tubule cell line, RPTEC/TERT1. Toxicology and applied pharmacology, 375, 5-16.

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