Electron microscopy : use of lectin-gold after embedding.

Fixation of tissues for postembedding gold-lectin histochemistry calls for a fixative that preserves the ultrastructure of the tissue, has no inhibiting effect on lectin binding sites, and binds the glycoconjugates, which carry these lectin binding sites within the tissue so strongly that they cannot be washed out during the dehydration and ensuing embedding steps. There is no "one and only" fixative that has all these properties, and every fixative used for postembedding gold-lectin histochemistry must be viewed, at best, as a compromise. One common fixative, more or less exhibiting the properties required, is a mixture of 4% formaldehyde and 0.5% glutaraldehyde in phosphate buffer at pH 7 35. The use of osmium tetroxide as a fixative must, in general, be avoided for postembedding lectin histochemistry since it disturbs the reactivity of lectin binding sites.

Light and electron microscopic localization of the alpha 1-chain and the E1 and E8 domains of laminin-1 in mouse kidney using monoclonal antibodies to establish the orientation of laminin-1 within basement membranes.

To localize the different domains of the laminin-1 molecule in tissues and gain insight into their in vivo relevance, we raised rat anti-mouse monoclonal antibodies (MAbs) against the entire molecule. Then we tested eight of the 20 clones producing anti-laminin-1 MAbs to specify their reactivity towards the alpha 1-, beta 1-, and gamma 1-chains and the elastase-cleaved fragments of the laminin-1 molecule. We found three MAbs with high titers in ELISA that showed good reactivity in embedded tissue. One of these reacted specifically against the E1 fragment, one against the E8 fragment, and one MAb detected the alpha 1-chain of laminin-1 but not the beta 1- or gamma 1-chain. All three MAbs are useful for light immunohistochemical investigations on cryosections and on paraffin-embedded material, and for ultrastructural localization of laminin-1 in LR Gold-embedded mouse tissue. Antibody staining of the E1 and E8 domains of laminin-1 revealed distinct localization of the molecule in the proximal tubule basement membranes of mouse kidney. The short arms (E1) of the laminin-1 molecule are predominantly located in the lamina lucida and the long arms (E8) are oriented towards the lamina fibroreticularis. Therefore, both MAbs are useful for studies of the orientation of the laminin-1 molecule in basement membranes. The distal tubule basement membranes did not show any distinct pattern of laminin-1 distribution. In general, the distal tubules showed the strongest reactions over the entire width of the basement membrane for all three MAbs. In contrast, the proximal tubule basement membranes showed somewhat weaker reactivity but a distinct pattern of laminin-1 distribution, with the E1 fragments oriented towards the adjacent epithelial cell surface.

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney.

The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques. After preembedding immunostaining for laminin using IgG--PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A--PO, staining of the 1. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the 1. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the 1. fibroreticularis and the 1. rara but not in the 1. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.