Hypoxia was determined by the Hypoxyprobe Kit (Catalog#HP1, Burlington, MA, USA). Pimonidazole forms adducts with thiol-containing proteins if oxygen level in cell is below 14 µM. In short, MCTS were treated with 100 µM pimonidazole HCl for 2 hours. The MCTS were then collected, washed once in PBS, and then fixed with paraformaldehyde for 1 hour and then stored in 70% ethanol until embedding in paraffin. Sections were cut in a microtome and stained with a mouse antibody toward pimonidazole (Mab1, 60 µg/mL, diluted 1:100), a secondary anti-mouse antibody (Vector Laboratories) and visualized with DAB.
Secondary anti mouse antibody
Manufactured by Vector Laboratories
The secondary anti-mouse antibody is a laboratory reagent used in immunoassays and other immunological techniques. It is designed to bind to primary mouse antibodies, allowing for the detection and visualization of target molecules in a sample.
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Lab products found in correlation
2 protocols using secondary anti mouse antibody
1
Hypoxia Detection in MCTS
2
Immunohistochemical Evaluation of S100A7 in Tumors
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Tumor sections (4 µm) were cut and deparaffinized in Xylene and rehydrated in graded ethanol. Heat-induced antigen retrieval was performed with citric acid buffer (pH6) using a microwave oven for 10 min, which was followed by H2O2 treatment in order to block endogenous peroxidase. The slides were then incubated with horse serum, followed by incubation with primary antibody (mouse mAb S100A7 dilution 1:100, clone 47C1068; Santa Cruz Biotechnology) overnight. Secondary anti-mouse antibody (1:200; Vector Laboratories, Inc.) was then added followed by the ABC kit (Vectastain; Vector Laboratories, Inc.). The staining was developed in DAB followed by hematoxylin counterstaining. The staining was evaluated by three researchers blinded for clinical outcome (LH, DL and AN). The percentage of positive tumor cells per total tumor cells was evaluated for each section and a cut-off value of 30% (more specifically, the proportion of immunostained cells, irrespective of whether the staining was cytoplasmic or nuclear) was applied as previously described, and since we here wanted to compare out data to such studies by others (15 (link)).
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