Hi Everyone,
I am trying to label mouse vasculature using Lectin Dylight 594 by transcardial perfusion. I have had success at 20ug/mL with the labeling and the architecture of the vasculature looks great when I frozen section directly onto the slide, coverslip, and take it to the scope. However, I need to stain these sections with a Th antibody and historically I have done this for IF using Free Floating sections. When I try to stain this way with the Lectin perfused tissue, the staining is quite messy, bleeds through to other channels, and has high background...
Is Lectin perfusion compatible with Free-Floating IF staining afterwards? Would I be better off frozen sectioning onto the slide and then staining (this is difficult because I get a lot of chatter/folding and I need to maintain accurate spatial resolution)? Does anyone have any experience using paraffin embedding and then IF staining after the Lectin perfusion?
Thank you!
Lectin perfusion followed by free-floating immunofluorescence (IF) staining can be a challenging technique, as you have observed. Lectins are known to bind to various glycoproteins and glycolipids, and their binding can interfere with subsequent antibody labeling during IF staining. However, there are strategies you can try to optimize your staining protocol.
1. Blocking: Increase the blocking step before applying the primary antibody. Use a blocking solution containing serum or other blocking agents to minimize non-specific binding of lectins and reduce background staining.
2. Titration: Optimize the concentration of lectin and primary antibody. Try different concentrations of lectin during perfusion and titrate the primary antibody concentration during the IF staining step to find the optimal conditions.
3. Sequential staining: Perform lectin staining and IF staining sequentially on the same tissue section. After completing the lectin staining protocol, quench the lectin fluorescence using a blocking solution or a glycine-based buffer. Then proceed with the IF staining using the Th antibody.
4. Washes: Increase the number and duration of washes between lectin staining and IF staining steps. This will help to remove any unbound lectin and reduce background staining.
5. Secondary antibody selection: Use secondary antibodies with minimal cross-reactivity to lectins. Ensure that the secondary antibody used in the IF staining step does not bind to lectins or cross-react with lectin-Dylight 594.
Regarding your question about frozen sectioning versus paraffin embedding, frozen sections generally provide better preservation of antigenicity and spatial resolution compared to paraffin-embedded sections. However, if you are experiencing issues with frozen sections such as chatter or folding, you can explore paraffin embedding as an alternative. Paraffin sections may require additional antigen retrieval steps to unmask epitopes for effective antibody binding.
It is important to note that these suggestions are general guidelines, and optimization may still be necessary for your specific experimental conditions and reagents.
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