In the setting of
stroke,
ischemia not only impairs neuronal function, but also detrimentally affects the different components of the neurovascular unit, which are shown to be involved in the transition from reversible to long-lasting tissue damage. In this context, the glial
proteins myelin basic
protein (MBP) and the 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) as well as the vasculature-associated basement membrane
proteins laminin and
collagen IV have been identified as
ischemia-sensitive elements. However, available data from immunofluorescence and Western blot analyses are often found to be contradictory, which renders interpretation of the respective data rather difficult. Therefore, the present study investigates the impact of tissue pre-treatment and antibody clonality on immunofluorescence measurements of the mentioned
proteins in a highly reproducible model of permanent
middle cerebral artery occlusion. Here, immunofluorescence labeling using polyclonal
antibodies revealed an increased immunofluorescence intensity of MBP, CNP,
laminin and
collagen IV in ischemic areas, although Western blot analyses did not reveal increased
protein levels. Importantly, contrary to polyclonal
antibodies, monoclonal ones did not provide increased fluorescence intensities in ischemic areas. Further, we were able to demonstrate that different ways of tissue pre-treatment including
paraformaldehyde fixation and
antigen retrieval may not only impact on fluorescence intensity measurements in general, but rather one-sidedly affect either ischemic or unaffected tissue. Therefore, immunofluorescence intensity measurements do not necessarily correlate with the actual
protein levels, especially in
ischemia-affected tissue and should always be complemented by different techniques to enhance reproducibility and to hopefully overcome the translational roadblock from bench to bedside.