HE染色及免疫组化英文步骤.docx

组织名称 were immersed in 4% paraformaldehyde for 4h, and transfered to 70% ethanol. Individual lobes of 组织名称 biopsy material were placed in processing cassettes, dehydrated through a serial alcohol gradient, and embedded in paraffin wax blocks. Before immunostaining, 5-um-thick lung tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. And then stained with hematoxylin and eosin (H&E). After staining, sections were dehydrated through increasing concentrations of ethanol and xylene.Immunohistochemistry for CCR7, CD206, and F4/80.Antigens were unmasked by microwaving sections in 10 mmol/L citrate buffer, pH 6.0 (15 minutes), and immunostaining was undertaken using the avidinbiotinylated enzyme complex method with antibodies against CCR7 at a concentration of 1ug/ml, CD206 at a concentration of 1 ug/ml, F4/80 at a concentration of 1 ug/ml, and equivalent concentrations of polyclonal nonimmune IgG controls. After incubation with the appropriate biotin-conjugated secondary antibody and subsequently with streptavidin solution, color development was performed using 3,3 diaminobenzidine tetrahydrochloride(Vector Laboratories) as a chromogen. Sections were counterstained using Gill-2 hematoxylin (Thermo-Shandon, Pittsburgh, PA). After staining, sections were dehydrated through increasing concentrations of ethanol and xylene.。