Immunohistological Detection of Pathogens in Brain Tissue

Take a formalin-fixed human brain tissue embedded in paraffin. The tissue exhibits pathogenic microbes present both inside the cells and extracellularly.

Wash to remove the paraffin, then rehydrate the tissue using decreasing alcohol concentrations.

Heat with an acidic buffer to break the formalin-induced protein crosslinks, unmasking antigenic epitopes on the bacteria.

Incubate with ammonium chloride to neutralize residual reactive aldehyde groups formed during fixation, preventing non-specific interaction with antibodies.

Treat with a detergent to permeabilize cellular membranes, facilitating antibody penetration.

Apply a blocking solution to prevent non-specific antibody binding.

Incubate with primary antibodies that target extracellular and intracellular microbes.

Apply fluorophore-conjugated secondary antibodies that bind to the primary antibodies.

Add a fluorescent DNA-binding dye to stain the nuclei.

Treat with an autofluorescence eliminator to quench fluorescence from autofluorescent intracellular granules, minimizing background signals.

Using fluorescence microscopy, examine the tissue to confirm the presence of microbes.