Browsing by Subject "Antigen retrieval"

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    An effective and practical immunohistochemical protocol for bone specimens characterized by hyaluronidase and pepsin predigestion combined with alkaline phosphatase-mediated chromogenic detection
    (F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2015) Li, Shangfu; Liu, Bin; Tian, Ming; Zhang, Liangming; Tickner, Jennifer; Xu, Jiake; Rong, Limin
    The aim of this study was to provide an effective procedure for immunohistochemistry (IHC) investigations of bone specimens. Samples from rat femoral and human vertebral bone were processed with a detailed and effective IHC protocol summarized here. First, a novel antigen retrieval (AR) method of hyaluronidase combined pepsin predigestion (H+P) was established and the optimal concentration and pH value for AR of bone specimens were determined. Second, the newly developed method was compared with existing AR methods (boiling in sodium citrate, hyaluronidase predigestion (H) and pepsin predigestion (P), with PBS only as the negative control) using two chromogenic detection systems (horseradish peroxidase (HRP) and alkaline phosphatase (AP)) to evaluate their efficacy in obtaining the best IHC results for bone samples. Considering the drawbacks of significant shrinking and detachment from slide for heat retrieval methods and the only moderate immunolabeling for H and P, H+P was the optimal AR method for IHC of bone specimens with the advantages of both good morphological preservation and strong immunoreactivity. Moreover, AP-mediated chromogenic detection was superior to HRP-labeled chromogenic detection due to significantly less nonspecific staining. In conclusion, we presented an effective and practical IHC protocol for bone specimens characterized by H+P predigestion combined with APmediated chromogenic detection. Finally, a detailed troubleshooting guide was provided for common mistakes that occur during IHC processing of the bone tissue samples.
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    Antigen retrieval on epoxy sections based on tissue infiltration with a moderately increased amount of accelerator to detect immune complex deposits in glomerular tissue
    (Murcia : F. Hernández, 1999) Brorson, S.H.; Andersen, T.; Haug, S.; Kristiansen, I.; Risstubben, A.; Tchou, H.; Ulstein, J.
    We wanted to examine the effect of antigen retrieval on epoxy sections where the tissue had been infiltrated by resin containing moderately increased amounts of accelerator. The concentration of accelerator DMP-30 (Tri(Dimethy1 Amino Methyl) Phenol) was varied in the range of 0% to 4% in the infiltration step of the tissue processing. Some of the epoxy sections were fixed in osmium tetroxide, and for others this fixative was avoided. Immunogold labeling was performed on epoxy sections and LR-White sections of renal tissue with IgG-deposits, and the antibody used was anti-IgG. Antigen retrieval was performed by heating the sections in citrate buffer. The amount of immunogold labeling on retrieved sections increased according to the amount of accelerator the non-osmicated epoxy sections were based on in the infiltration steps. For the osmicated epoxy sections these differences were less pronounced. The immunogold labeling of retrieved epoxy sections was up to 70% of LR-White labeling. In addition to breaking fixation bond introduced by the chemical fixation, we believe that the antigen retrieval also breaks bonds between the epoxy resin and the embedded tissue. The combination of increased amount of accelerator in the tissue infiltration and antigen retrieval by heating the sections in citrate buffer is a good method for improving the immunolabeling of epoxy sections.
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    Heat-induced antigen retrieval of epoxy sections for electron microscopy
    (Murcia : F. Hernández, 2001) Brorson, S.H.
    The purpose of this manuscript is to review the literature on the use of heat-induced antigen retrieval methods to enhance the immunolabeling of epoxy sections at the electron microscopical level. The history of the development of antigen retrieval by heating formaldehyde fixed paraffin sections in a buffer solution is given in short, and how this technique has been extended to resin sections and in particular epoxy sections is explained. Theories for the mechanism of enhancement of the immunolabeling of epoxy sections by the heat-retrieval method are discussed, and it is finally speculated whether most of the mechanisms for antigen retrieval on epoxy sections in heated buffer solution are essentially the same as for conventional immunoenhancing by deplastizing and etching. The more accelerator used in the processing of the tissue the more intense the immunolabeling of the heated epoxy sections becomes. The intensity of immunolabeling of the epoxy sections increases with the temperature in the heated buffer solution, and the intensity is significantly higher at high autoclave temperatures than at 95 OC, Heat-induced antigen retrieval is also compared with other, conventional techniques for enhancing the immunolabeling of epoxy sections.