Cytochemical studies of the intracellular processing of glycoconjugates

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1)The analysis of glycosylation using postembedding staining method.Cis side of the Golgi apparatus was labeled with DBA, HPA and SBA, trans side and secretory granules were stained with HPA, PNA and RCA-I, UEA-I and LFA labeled transmost Golgi apparatus. By this method, the process of glycosylation of the Golgi apparatus was clearly analyzed.2)The analysis of glycosylation using preembedding staining method.MPA labeled perinuclear space, inner lumen of rough endoplasmic reticulum and cis side of the Golgi apparatus. DBA stained the same region of the Golgi apparatus. PNA labeled the intermediate and RCA-1, UEA-1 and LFA stained the trans side of the Golgi apparatus. Secretory granules were labeled with RCA-1, UEA-1 and LFA. The results-obtained with these two methods were not inconsistent.3)The analysis of glycosylation process using monoclonal antibodies to ABO substances.Anti A labeled the whole lamellae of the Golgi apparatus, while anti B and anti H labeled the trans side of the Golgi apparatus. The difference of the labeling was noticed among antibodies.4)The development of a new cytochemical method to stain acidic glycoconjugates.By making cationic colloidal gold with poly-L-lysine, changing pH of the staining solution and setting many control experiments, we established a new staining method of acidic glycoconjugates. This method is used both in light microscopic level and electron microscopic level. The sites of sialylation and sulfation were analyzed using this method and found that both localized at the trans side of the Golgi apparatus.5)The improvement of cytochemical demonstration method of sulfated glycoconjugates.By adding physical development step to high iron diaminethiocarbohydrazide-silver proteinate method, we succeeded in improving the staining sensitivity of this method. This method allows us to develop in the bright field condition. The site of sulfation has been analyzed and found to localize at the trans Golgi apparatus.