Effect of fixation protocols on in situ detection of L-selectin ligands

In situ binding of (chimeric) proteins to tissue sections is a widely used method to identify ligands and their localization. Many different protocols for the fixation of frozen tissue sections are used for in situ binding studies. We report the effects of different fixation protocols on the binding pattern observed using in situ binding of an L-selectin–IgM chimeric protein to both rat lymph node and kidney tissue sections. L-selectin is a C-type lectin, expressed on leukocytes and is involved in both lymphocyte homing and migration upon inflammation. We show that different in situ binding patterns in rat kidney are observed using different fixation protocols, including glutaraldehyde, methanol, formaldehyde and acetone fixation. The observed staining is specific, as it can be blocked in the presence of EGTA, an L-selectin blocking antibody or by ligand competition. Enzymatic pre-treatment of the tissue sections using sialidase, heparitinase I or chondroitinase ABC has differential effects on in situ binding depending on tissue type and fixation protocol. These data indicate that special attention should be paid in choosing a fixation protocol for in situ binding studies, especially when using lectins. This could prevent biologically relevant ligands remaining undetected or wrong conclusions being drawn based on the localization of observed binding.