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.
Miosge
N
N
Universität Göttingen, Abteilung Histologie, Germany.
Günther
E
E
Heyder
E
E
Manshausen
B
B
Herken
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D001485:Basement Membrane / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000737:chemistry*; D007797:Laminin / Q000032:analysis*; D051379:Mice; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000032:analysis*
D000911:Antibodies, Monoclonal; D007797:Laminin; D010446:Peptide Fragments