Effects of different antigen retrieval methods and fixative pH values on immunostaining of mouse incised wound skin / 中国法医学杂志

ABSTRACT

Objective To evaluate the influence of different antigen retrieval methods and pH values of fixatives on immunostaining of incised wound skin.Methods 3d-post-incision mouse skin were sampled and fixed in 4% formalin buffered with PBS at pH 5.0,6.0,7.0,8.0 and 9.0.The deparaffinized sections were treated 0.01mmol citrate buffuer(pH 6.0)in high pressure cooker or by microwave heating,or with pepsin,trypsin or microwave+pepsin.Thereafter,immunostainings of caspase-3,caspase-6,caspase-7,IL-8,and IL-10 were evaluated by immunohistochemical SABC method with ratio of immunostaining-positive cells,respectively.Results The immunostaining-positive cell ratios in sections treated by heating retrieval was higher than by proteinase treatment.The positive cell ratio was slightly higher by high pressure than by microwave heating.Sections were more easier peeled off during high pressure treatment than during micrwave heating.Sections treated with pepsin were fallen off easier than with trypsin digestion.The highest positive cell ratio was detected in sections by microwave+trypsin treatment without section peeling off among all antigen treatment methods.The best result was obtained in sections fixed in buffered formalin at pH 7.0 and 8.0.Conclusion The best immunostaining result may be obtained in mouse incised wound sections fixed in buffered formalin at pH 7.0 and 8.0 with microwave+trypsin treatment.