ABSTRACT
BACKGROUND: Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. NEW METHOD: To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. COMPARISON WITH EXISTING METHOD AND RESULTS: An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. CONCLUSION: Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM.
Subject(s)
Antibodies/chemistry , Immunohistochemistry/methods , Microscopy, Electron/methods , Neurons/ultrastructure , Tissue Embedding/methods , Tissue Fixation/methods , Aldehydes/chemistry , Aldehydes/immunology , Animals , Glutaral/chemistry , Glutaral/immunology , Hippocampus/ultrastructure , Male , Primary Cell Culture , Rabbits , Rats, WistarABSTRACT
In the original publication of the article the author name M. Schupps was incorrect.
ABSTRACT
The SNARE protein SNAP-25 is well documented as regulator of presynaptic vesicle exocytosis. Increasing evidence suggests roles for SNARE proteins in postsynaptic trafficking of glutamate receptors as a basic mechanism in synaptic plasticity. Despite these indications, detailed quantitative subsynaptic localization studies of SNAP-25 have never been performed. Here, we provide novel electron microscopic data of SNAP-25 localization in postsynaptic spines. In addition to its expected presynaptic localization, we show that the protein is also present in the postsynaptic density (PSD), the postsynaptic lateral membrane and on small vesicles in the postsynaptic cytoplasm. We further investigated possible changes in synaptic SNAP-25 protein expression after hippocampal long-term potentiation (LTP). Quantitative analysis of immunogold-labeled electron microscopy sections did not show statistically significant changes of SNAP-25 gold particle densities 1 h after LTP induction, indicating that local trafficking of SNAP-25 does not play a role in the early phases of LTP. However, the strong expression of SNAP-25 in postsynaptic plasma membranes suggests a function of the protein in postsynaptic vesicle exocytosis and a possible role in hippocampal synaptic plasticity.
