Evaluation of the antigen retrieval method for immunofluorescence staining of type Ⅳ collagen in paraffin-embedded skin tissues
Objective To explore the best antigen retrieval method for immunofluorescence staining of type Ⅳ collagen in paraffin-embedded(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 type Ⅳ collagen 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 high-pressure 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 type Ⅳ collagen in paraffin-embedded skin tissues.
Skin tissuesImmunofluorescence in paraffin-embedded tiuusesAntigen retrievalType Ⅳ collagen
万方数据
维普
