皮肤石蜡组织Ⅳ型胶原免疫荧光抗原修复方法的评估

Evaluation of the antigen retrieval method for immunofluorescence staining of typeⅣcollagen in paraffin-embedded skin tissues

目的 寻找皮肤石蜡包埋组织Ⅳ型胶原免疫荧光(IF-PE)最佳抗原修复方法。方法以2021年1月至2022年12月在上海交通大学医学院附属儿童医院确诊的X-连锁Alport综合征患儿和父(母)亲共14例皮肤组织,冰冻组织免疫荧光(IF-FT)为自身对照,比较不同抗原修复法IF-PE染色部位和强度。结果 与Ⅳ型胶原IF-FT自身对照比较,采用蛋白酶K修复,仅部分患儿基膜正常分布(6/14),而角质层均异常表达;微波、高温水浴和高压蒸汽3种修复方法,基膜均无正常分布,角质层异常分布(分别为2/14,11/14,12/14);微波联合酶修复法仅2例基膜正常分布,未见角质层异常表达;高压蒸汽联合酶和高温水浴联合酶2种修复法基膜全部正常分布,角质层异常表达(分别为14/14和2/14),表达强度两者接近。结论高温水浴联合蛋白酶K可作为皮肤石蜡包埋组织Ⅳ型胶原免疫荧光理想的抗原修复法。

Objective To explore the best antigen retrieval method for immunofluorescence staining of typeⅣcollagen in paraffinembedded(IF-PE)skin tissues.Methods The skin tissues were collected from 14 individuals in this study,including children with X-linked Alport syndrome and their parents,from Shanghai Children's Hospital between January 2019 and December 2022.The staining localization and intensity of typeⅣcollagen IF-PE with different antigen retrieval methods were compared and the immunofluorescence on frozen tissues(IF-FT)was used as self-control.Results IF-PE showed that,in comparison to the self-control,the protease K antigen retrieval method resulted in only a fraction of skin tissues(6/14)exhibiting normal basal membrane distribution,while the abnormal stratum corneum expression was observed in all skin tissues There were no normal distribution of basement membrane was observed in skin tissues through three antigen retrieval methods including microwave method,hyperthermic water bath method and high-pressure steam method.The microwave method resulted in abnormal stratum corneum expression in 2/14 cases,while this occurred in 11/14 cases with the hyperthermic water bath and in 12/14 case with the high-pressure steam method.The normal distribution of basal membrane was only observed in 2 cases by microwave combined in combination with protease K antigen retrieval method,and no abnormal expression was found in stratum corneum.The normal distribution of basal membrane of 14 cases were observed by the two antigen retrieval methods,including highpressure steam combined with protease K and hyperthermic water bath combined with protease K.The high-pressure steam combined with protease K method resulted in abnormal stratum corneum expression in 14/14 cases,while this occurred in 2/14 cases with the hyperthermic water bath combined with protease K method;However,the similar expression intensity were observed in both methods.Conclusions The combination of hyperthermic water bath and protease K represents an optimal approach for immunofluorescence staining of typeⅣcollagen in paraffin-embedded skin tissues.