Procedure for Light Microscopy

Human brain tissues, for example, are fixed postmortem with 10% formalin for 24-48 hr, dehydrated, and embedded in paraffin. Sections, cut with a rotary microtome, are mounted on coated glass slides. The sections are rinsed three times for 5 min each with 0.1 M sodium phosphate buffer (pH 7.4) and then transferred to 10-15 mM sodium citrate 

The sections are incubated in a primary antibody (diluted appropriately) for 72 hr at 4°C in a sealed humid chamber the incubation is carried out by applying droplets of the antibody to the sections. After being rinsed in the buffer, the sections are incubated for 90min in secondary antiserum diluted 1 50 with PBX (0.3% Triton X-100, 0.01% sodium azide and 0.1 M phosphate buffer) and then treated for 1 hr under agitation in peroxidase-antiperoxidase (PAP), diluted 1 100 with PBX, in a sealed humid chamber in both cases. 

For assessing the antigen retrieval effectiveness, the staining intensities observed under the microscope can be divided into five grades + + + +,+ + +,+ +,+ or- for 

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Immunohistochemical Procedures for Light Microscopy The immunohistochemical staining using a biotin-streptavidin-peroxidase complex was performed according to the... 

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