FFPE tumor sections (or tumor microarray (TMA) slides) were melted at 70 °C for 30 min, de-paraffinized using xylene and rehydrated in serial alcohol washes. The slides were pressure cooked at 15 psi for 15 min in 1× DAKO antigen-retrieval buffer (Agilent Technologies). Slides were subjected to two 5-min standing washes in PBS before blocking in 1× Carbo-Free Blocking Solution (Vector Laboratories) for 2–3 h, followed by two more washes and staining with the indicated lectin and/or antibodies before washing and mounting with VECTASHIELD and DAPI (Vector Laboratories).
Dako antigen retrieval buffer
Manufactured by Agilent Technologies
The DAKO antigen-retrieval buffer is a laboratory reagent used to facilitate the exposure of antigenic sites within tissue samples prior to immunohistochemical staining. The buffer is designed to help unmask or retrieve antigens that may be obscured or modified during the sample preparation process.
Automatically generated - may contain errors
Lab products found in correlation
Carbo free blocking solution, by Vector Laboratories (1 mentions)
Axiocam hrc camera, by Zeiss (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Orb182397, by Biorbyt (1 mentions)
Axioskop light microscope, by Zeiss (1 mentions)
Ba 5000, by Vector Laboratories (1 mentions)
Bx51 light microscope, by Olympus (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
4 protocols using dako antigen retrieval buffer
1
Immunohistochemical Staining of FFPE Tissue Sections
2
Immunohistochemical Detection of tdTomato+ ILC2s
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For staining of tdTomato-positive ILC2s in Red5 mice, 2.5-μm-thick paraffin sections from kidney and SILP were heated at 98°C for 40 min in DAKO antigen retrieval buffer (pH 9; Agilent Technologies). Unspecific binding was blocked with 5% horse serum (Vector Laboratories) and 0.05% triton-X100 in PBS. The sections were then incubated with goat anti-tdTomato (1:2,000, orb182397; Biorbyt) in 5% horse serum overnight at 4°C. Next, the sections were incubated with biotinylated rabbit anti-goat antibody (BA-5000; Vector Laboratories) followed by incubation with anti-rabbit polymer (POLAP-006; Zytomed Systems). Finally, the development and nuclear staining of stained sections were performed with New Fuchsin and Hemalaun, respectively. Tissue slices were evaluated with an Axioskop light microscope (Zeiss) and photographed with an Axiocam HRc camera (Zeiss).
3
Immunohistochemical Analysis of Intestinal GUCY2C and GUCA2A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following deparaffinization and rehydration in a xylene-ethanol-water gradient, 4μm sections of wild-type mouse [C57BL/6(J)] intestines underwent antigen retrieval by heating at 100°C for 15 minutes in a pressure cooker in pH 9.0 DAKO antigen retrieval buffer (Agilent Technologies, Santa Clara, CA; S236784-2). The monoclonal antibody to GUCY2C (MS20) was described previously,20 (link) and the antisera to GUCA2A (#2538) was kindly provided by Dr. M. Goy.33 (link) GUCY2C and GUCA2A were detected by tyramine signal amplification, as described previously.53 (link) Images were captured using an EVOS FL auto cell imaging system (Thermo Fisher Scientific).
4
Quantification of Glomerular CD45+ Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed paraffin-embedded kidney sections (3 µm) underwent antigen-retrieval in DAKO antigen-retrieval buffer (Agilent). Sections were blocked with PBS/1% BSA, followed by incubation with rat anti-mouse CD45-biotin, then anti-rat-HRP secondary antibody (both in PBS/0.2% BSA). Chromogenic substrate (DAKO diaminobenzidine chromogen solution; Agilent) was added to sections followed by counter-staining with hematoxylin and coverslip mounting with DPX (Sigma-Aldrich). Sections were imaged using an Olympus BX-51 light microscope at 20x objective magnification (Olympus Australia), capturing at least six images of each kidney cortex. Images were analyzed using ImageJ software by isolating the red channel and setting the color threshold to 120 to visualize punctate staining. CD45+ staining was quantified using the polygon tool to trace each glomerulus and measuring the area fraction. A minimum of 30 glomeruli were analyzed per kidney, with the mean taken to represent each sample.
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!