The tumors were fixed in 10% neutral buffered formalin and included in paraffin. 4μm-thick tissue sections obtained from paraffin embedded tissues were deparaffinized, rehydrated and stained with H&E to define tumor histotypes. Immunohistochemistry was performed using a polymer detection method. Briefly, the antigen retrieval was performed using Novocastra Epitope Retrieval Solution pH 9 in a PT Link Dako pre-treatment module at 98°C for 30 minutes. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% H2O2 and Fc blocking by a specific protein block (Novocastra UK), the samples were incubated with primary antibodies. For rat anti-mouse monoclonal CD8a (4SM15) or CD4 (4SM95) 1:100 pH9 (eBioscience) antibodies, the staining was revealed using goat anti-rat IgG (H&L) specific secondary antibody 1:500 (Novex by Life Technologies) and AEC (3-Amino-9-ethylcarbazole) substrate-chromogen. Slides were analyzed under a Zeiss Axioscope A1 and microphotographs were collected using a Zeiss Axiocam 503 Color with the Zen 2.0 Software (Zeiss). Quantitative IHC data for CD8 and CD4 marker was calculated by counting the number of CD8+ or CD4+ cells in five fields at 40X magnification.
Epitope retrieval solution ph 9
Manufactured by Agilent Technologies
Epitope Retrieval Solution pH 9 is a laboratory product designed for use in immunohistochemistry (IHC) procedures. It is a solution with a pH of 9 that is used to unmask or retrieve antigenic epitopes on tissue samples, which can improve the detection and visualization of target proteins during IHC analysis.
Automatically generated - may contain errors
2 protocols using epitope retrieval solution ph 9
1
Immunohistochemical Analysis of Tumor-Infiltrating T Cells
2
MGMT Immunohistochemistry in FFPE Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC was performed on 4 µm sections of FFPE tumor blocks. Slides were then deparaffinized in xylene and rehydrated through graded alcohols. Antigen retrieval was performed in Epitope Retrieval Solution pH 9 (×10 concentration—Dako system) at 110 °C for 10 min in TBS and endogenous peroxidase was inactivated with 3% hydrogen peroxide. Slides were incubated with mouse monoclonal MGMT primary antibody (MT 3.1, Invitrogen, Waltham, MA, USA) used at a final dilution of 1:250 for 1 h after protein blocking (BSA 5% in PBS 1×). Diaminobenzidine, as a chromogenic substrate, was used to visualize immunoreactivity. Finally, the sections were lightly counterstained with hematoxylin and mounted. One pathologist, blinded to methylation pattern and other parameters, evaluated and scored MGMT expression in the tumor sections. Staining for MGMT protein was considered to be positive if the MGMT staining was evenly distributed in the cell nuclei. Negative staining was defined as staining restricted to the cytoplasm and granular nuclear reactivity.
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!