00000ctm a22000001a 4500 UP-1685523046125399757 Buklod 20230503092606.0 a r |||| u| ta 071031s xx d r |||| u| (iLib)UPMNL-00000053240 UPM Med LG 995 2005 B3 G85 Guillergan, Fred P. . Effectiveness of three antigen retrieval techniques for use in immunostaining of formalin-fixed schistosoma japonicum egg antigens in mouse granulomatous livers Fred P. Guillergan. 61 leaves. Thesis (MS Biochemistry)--University of the Philippines Manila Antigen retrieval systems have expanded the scope of immunohistochemical studies. Various antigen-unmasking techniques were developed and utilized as pretreatment for formalin-fixed paraffin embedded tissues prior to immunological studies. Antigen retrieval systems are necessary for immunostaining of formalin-fixed specimens because it uncovers the epitopes masked by hydroxy-methylene bridges that cross-links macromolecules in and outside the cells.In this study, effectiveness of antigen retrieval systems was tested for immunohistochemistry studies. Schistosoma japonicum-infected mice gradulomatous liver tissues fixed in 10% neutral buffered formalin at either 20-hour or 30-day fixation periods were utilized. Effect of both fixation times on immunostaining was evaluated.Four procedures were used in this study, three for antigen retrieval systems and one for no antigen retrieval. The three methods employed as antigen retrieval prior to immunostaining included, (1) heat-introduced antigen retrieval (2) proteolytic enzyme digestion, and (3) combination of heat-introduced and enzyme digestion. For the heat-induced antigen retrieval, water bath was utilized to incubate specimens at 100 percent C for 20 minutes. Proteolyic enzyme digestion was done with 0.1% trypsin at 37 degrees C for 30 minutes. The third antigen retrieval system was a combination of heat-induced and enzyme digestion methods. The same procedure for heat-induced antigen retrieval was applied to the combination treatment but was followed immediately after by enzyme digestion with 0.1% trypsin for 15 minutes.Citrate buffer was used as the antigen retrieval solution. Polyclonal antibody from serum of infected mice was utilized as primary antibody. Immunohistochemistry was done using DAKO LSAB 2 kits, and the procedure was followed as provided by the kit. The observers composed of two pathologists and the researcher interpreted the immunostaining results. The readers were blinded during microscopic evaluation of the immunostained slides. The results of the study showed that antigen retrieval is necessary for immunohistochemistry on formalin-fixed S. japonicum egg antigens. The three AR systems were effective in unmasking epitopes based on the three readings done by the observers. The most sensitive ARS in 20-hour fixed specimens is Enzyme digestion with 80% sensitivity. Statistical analysis using Pearson Chi-Square test showed no significant difference in immunohistochemistry among the antigen retrieval systems. The Mann-Whitney test showed no significant difference in immunostaining between tissues fixed for 20 hours and 30 days. However, sensitivity of enzyme digestion decreased from 80% to 73%. Specimens fixed with formalin for more than 48 hours up to 30 days can be utilized for immunohistochemistry studies using any of the antigen retrieval systems described. The use of 10% neutral buffered formalin for S. japonicum egg antigen was established after successful immunostaining results. Future studies whether for diagnostic or research purposes involving S. japonicum egg antigens can be guided by using the information gained from this research papers. Immunohistochemistry. Antigens Analysis. Pathology. Methods. UPMNL MED LG 995 2005 B3 G85 Thesis