To begin dewaxing previously prepared paraffin sections of the Peyer's patch isolated from immunoglobulin A or IgA neuropathy model mouse, submerge each section sequentially in xylene and ethanol gradients for five minutes. Then, rinse the section in distilled water. For antigen retrieval, heat sodium citrate solution for two minutes in an autoclave.
Then, place the tissue slice in the solution and heat it at a high temperature for five minutes. Once cooled down, incubate the section in a 3%hydrogen peroxide solution for 15 minutes at room temperature away from light. Then, apply 10%goat serum evenly on the tissue section and incubate for 30 minutes at room temperature to block the section.
After shaking off the blocking solution, apply an appropriate amount of the prepared primary antibody to each section. Incubate the sections laid flat in a wet box overnight at four degrees Celsius. On the next day, cover the tissue with a drop of the HRP-labeled secondary antibody and incubate it at room temperature for 50 minutes.
Then, apply freshly prepared DAB chromogenic solution to each section and wait til color development. Restain the section with hematoxylin for approximately one minute. Subsequently rinse the sections for 10 minutes with tap water until they return to a blue color.
For dehydration, place each section sequentially in gradients of ethanol and xylene for five minutes each. Once all the sections are dried slightly, seal each section with neutral gum. Immunohistochemical results showed that B-cell markers CD20 and CXCR5 expression were significantly higher in the IgA neuropathy model group, compared with the control group.
However, dioscin could inhibit the expression of the molecular markers.
