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.

Abstract

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 al-, PI-, and yl-chains and the elastase-cleaved fragments of the laminin-l molecule. We found three MAbs with high titers in ELISA that showed good reactivity in embedded tissue. One of these reacted specifically against the El fragment, one against the ES fragment, and one MAb detected the al-chain of laminin-1 but not the PI-or ylchain. AU three MAbs are useful for light immunohistochemical investigations on a y d o n s and on paraffra-embedded material, and for ultrastructural localization of laminin-l in LR Gold-embedded mouse tissue. Antibody staining of the El and ES domains of laminin-1 revealed distinct localization of the molecule in the proximal tubule basement mem-

Ultrastructural localization of microfibrillar fibulin-1 and fibulin-2 during heart development indicates a switch in molecular associations

“…After rinsing with PBS, the secondary gold-labelled goat-antirabbit immunoglobulin G (IgG) antibody (Medac, Hamburg, Germany), diluted 1:200 with PBS, was applied for 30 min at RT. The gold labelling of the antibody was described in detail in a previous investigation [36]. Reactions with antibodies against fibronectin and versican at a concentration of 1:200 were performed for 1 h, the N. Miosge et al…”

Basement membrane components are key players in specialized extracellular matrices

“…1c, d) of the murine kidney [44]. Furthermore, laminin-211 and -221 that are located in Schwann cells are deposited in mesh-like structures for organizing the receptors and cytoskeletal elements [45], whereas a fibrillar network, for example, of laminin-311 in the lung seems ideal for the transmission of mechanosignals such as stretch [46].…”

Ultrastructural colocalization of nidogen-1 and nidogen-2 with laminin-1 in murine kidney basement membranes

“…An affinity-purified rabbit antibody against recombinant nidogen-2 (0.4 mg/ml) was used and showed no crossreactivity with nidogen-1 . A monoclonal rat anti-mouse antibody was prepared against murine laminin-1, Engelbreth-Holm-Swarm (EHS) laminin, and reacted exclusively with the laminin α1 chain in immunoblots and is suitable for light and electron microscopic immunohistochemistry of laminin-1 (Miosge et al 1995(Miosge et al , 1999a. Ammonium sulfate precipitation resulted in IgG which was diluted in phosphate-buffered saline (PBS), pH 7.2, to a concentration of 1 mg/ml (Miosge et al 1995).…”

“…Pieces of renal cortex, 1 mm 2 in size, from adult NMRI mouse kidneys, EHS tumor, and day 7 and 17 mouse embryos were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde for 15 min, dehydrated in a graded series of ethanol up to 70%, and embedded in the acrylic resin LR gold (London Resin Company, Reading, UK). Semithin (1 µm) and ultrathin (0.08 µm) sections were cut following procedures already described in detail previously (Herken and Miosge 1991;Miosge et al 1995).…”