To validate the functionality of the chimeric adenoviruses, HPV16 E7 protein expression was analyzed 72 h post-transduction. For this purpose, the cells were resuspended in PreservCyt® (Cytyc Corp., Malborough, MA 01752, USA) Cytospins from 1 × 105 cells were prepared by centrifugation 500 rpm, 2 min in a CellSpin centrifuge (Tharmac, Wiesbaden, Germany). Staining procedures were done according to the protocol described by Lidqvist and colleagues [26 (link)]. Briefly, the cells were permeabilized in 0.3% Triton X-100 in TBS for 15 min at room temperature. Incubation with the mouse monoclonal antibody E716-41 (Fujirebio Diagnostics, Göteborg, Schweden) at a dilution of 1 µg/mL was performed overnight at 4 °C. After several washing steps, specific staining was visualized using the Dako REAL™EnVison™ kit (Dako, Jena, Germany), which uses 3,3’-Diaminobenzidine (DAB) as a chromogen. Slides were counterstained by hematoxylin and inspected using an Axioplan microscope (Zeiss, Jena, Germany).
Dako real envison kit
Manufactured by Agilent Technologies
Sourced in Germany
The Dako REAL EnVison kit is a ready-to-use, horseradish peroxidase (HRP)-based immunohistochemistry detection system. It is designed for the visualization of antigens in formalin-fixed, paraffin-embedded tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Hydrogen peroxide solution, by Agilent Technologies (3 mentions)
Protein block serum free, by Agilent Technologies (3 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (3 mentions)
Tissuefaxs workstation, by TissueGnostics (3 mentions)
Mayer s hematoxylin solution, by Agilent Technologies (2 mentions)
Anti mouse cd45 monoclonal antibody, by BD (2 mentions)
Axioplan microscope, by Zeiss (2 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
4 protocols using dako real envison kit
1
Analyzing HPV16 E7 Protein Expression
2
Quantifying CD45-Positive Cells in Kidney Tissue
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Formalin-fixed renal tissue sections were stained for CD45 using immunohistochemistry. Sections (4-µm-thick) were deparaffinized with xylene, rehydrated in a graded alcohol series, and then transferred to citrate buffer solution (pH 6.0). Slides were placed in a pressure cooker and heated by microwaving for 10 min to enhance antigen retrieval. After cooling, the kidney sections were immersed in a hydrogen peroxide solution (DAKO, Carpinteria, CA) for 30 min to block endogenous peroxidase activity, followed by overnight incubation at 4°C with serum-free protein block (DAKO). The next day, the slides were incubated with a 1:100 dilution of anti-mouse CD45 monoclonal antibody (BD Biosciences, San Jose, CA) for 1 h at room temperature. After being rinsed, the CD45-stained sections were incubated for 30 min at room temperature with a secondary antibody using a Dako REAL EnVison kit (DAKO). Subsequently, 3,3’-diaminobenzidine tetrahydrochloride (DAKO) was applied to the slides to produce a brown color and then the slides were counterstained with Mayer’s hematoxylin solution (DAKO).
To calculate the percentage of CD45-positive cells in kidney samples, whole fields of slides including both cortex and medulla were scanned and analyzed with a TissueFAXS work station (Tissue Gnostics, Vienna, Austria), as described previously (17 (link)).
To calculate the percentage of CD45-positive cells in kidney samples, whole fields of slides including both cortex and medulla were scanned and analyzed with a TissueFAXS work station (Tissue Gnostics, Vienna, Austria), as described previously (17 (link)).
3
Immunostaining for Renal CD45+ Cells
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Formalin-fixed renal tissue sections were immunostained for detection of CD45 as follows. Sections (4-μm-thick) were deparaffinized with xylene, rehydrated in a graded alcohol series, and placed in a citrate buffer solution (pH 6.0). Slides were placed in a pressure cooker and heated for 10 min to enhance antigen retrieval. After cooling, the kidney sections were immersed in a hydrogen peroxide solution (Dako, Carpinteria, CA) for 30 min to block endogenous peroxidase activity, followed by overnight incubation at 4°C with serum-free protein block (Dako). The next day, the slides were incubated with a 1:100 dilution of anti-mouse CD45 monoclonal antibody (BD Biosciences, San Jose, CA) for 1 h at room temperature. After being rinsed, the CD45-stained sections were incubated for 30 min at room temperature with a secondary antibody using a Dako REAL EnVison kit (Dako). Subsequently, 3,3′-diaminobenzidine tetrahydrochloride (Dako) was applied to the slides to produce a brown color, and the slides were counterstained with Mayer's hematoxylin solution (Dako).
A TissueFAXS workstation (Tissue Gnostics, Vienna, Austria) was used to analyze and calculate the percentage of CD45-positive cells in kidney samples, as described previously (18 (link)).
A TissueFAXS workstation (Tissue Gnostics, Vienna, Austria) was used to analyze and calculate the percentage of CD45-positive cells in kidney samples, as described previously (18 (link)).
4
Immunohistochemical Analysis of CD45 in Renal Tissue
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Immunohistochemical staining of a cluster of differentiation (CD) 45 (leukocyte common antigen) using formalin-fixed renal tissue sections was performed with reference to a previous report30 (link). The renal tissue sections (4-µm-thick) were deparaffinized using xylene, rehydrated in a graded alcohol series, and then transferred to citrate buffer solution (pH 6.0). The slides were placed in a pressure cooker and heated in microwave for 10 min to enhance antigen retrieval. After cooling, the slides were immersed in hydrogen peroxide solution (Dako, Carpinteria, CA) for 30 min to block the endogenous peroxidase activity. After overnight incubation at 4 °C with serum-free protein block (Dako), the slides were incubated at room temperature for 1 h with a 1:100 dilution of monoclonal rat anti-mouse antibody to CD45 (BD Biosciences, San Jose, CA). After being rinsed, the CD45-stained sections were incubated for 30 min with a secondary antibody using a Dako REAL EnVison kit (Dako) at room temperature. Staining with 3,3′-diaminobenzidine tetrahydrochloride (Dako) was performed on the slides to extract brown color, then counterstaining with Mayer's hematoxylin (Dako) was performed. A TissueFAXS workstation (Tissue Gnostics, Vienna, Austria) was used to analyze and calculate the percentages of CD45-positive cells infiltrated into renal tissues, as described previously30 (link).
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