This article describes a detailed protocol for immunohistochemical analysis of brain tumor neurospheres. The method involves paraffin embedding, sectioning, and staining to visualize tumor-specific biomarkers.
Take a slide with paraffin-embedded sections of brain tumor neurospheres, clusters of neural stem cells exhibiting tumor-specific nuclear protein biomarkers.
Heat to melt paraffin, remove it using xylene, and rehydrate the tissue using decreasing alcohol concentrations.
Heat in an acidic buffer containing detergent to unmask epitopes on the biomarkers and permeabilize cellular membranes.
Rinse, then treat with an oxidizing agent to inactivate endogenous peroxidase enzymes, preventing non-specific signals.
Apply a blocking solution to mask non-specific binding sites.
Incubate with primary antibodies targeting the biomarker epitopes.
Rinse, then treat with biotin-conjugated secondary antibodies that bind to the primary antibodies.
Rinse and incubate with a peroxidase enzyme-conjugated avidin, which binds to biotin.
Add a chromogenic substrate and an oxidizing agent.
Peroxidase utilizes the agent to convert the substrate into colored precipitates, labeling the biomarker location.
Counterstain the nuclei, dehydrate the tissue in alcohol, and mount the slide for imaging the biomarker distribution.
First, melt the paraffin by incubating the slides in a metal rack at 60 degrees Celsius for 20 minutes. Then deparaffinized the slides via incubation in a rehydration train at room temperature according to the Tex protocol. Store the slides in tap water to prevent nonspecific antibody binding. After this, incubate the slides in citrate buffer at 125 degrees Celsius for 30 seconds at 90 degrees Celsius for 10 seconds. Allow the slides to cool before rinsing them five times with distilled water.
Next, incubate the slides at room temperature in 0.3% hydrogen peroxide for 20 minutes. Then, wash the slides for five minutes three times in washing buffer with gentle agitation. Incubate the slides overnight in a humidified chamber set to 4 degrees Celsius with diluted primary antibody. After this, wash the slides three times for five minutes with gentle agitation.
Next, incubate the slides in diluted biotinylated secondary antibody at room temperature for an hour. Wash the slides three times for five minutes in washing buffer with gentle agitation. Then, incubate the slides in avidin biotinylated horseradish peroxidase complex at room temperature in the dark for an hour. Repeat the wash as previously described. After this, incubate the slides with dab hydrogen peroxide substrate bands for two to five minutes at room temperature.
Rinse the slides with tap water, and dip the slides in hematoxylin solution to counterstain them. Then rinse the slides thoroughly in tap water until the water runs clear. Dehydrate the slides according to the Tex protocol. Finally, coverslip the slides with a xylene-based mounting medium and allow them to dry on a flat surface at room temperature.
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