I am having difficulty getting specific binding with my rat spinal cord immunofluorescence. My protocol is as follows:
1. Remove spinal cord and quick freeze in TissueTek OCT.
2. Cut tissue on cryostat, laying slices directly on gelatin coated slides.
3. Acetone (-20 C) fixation for 10-20 minutes.
4. PBS wash twice, 10 minutes each.
5. Blocking for an hour in blocking buffer (5% FBS, 0.2% Triton X-100, PBS) at RT.
6. Drain blocking buffer and incubate primary antibody mixed in blocking buffer for 2 hours at RT (have also tried overnight at 4 C).
7. Rinse slides in PBS 3 times, 10 minutes each.
8. Incubate secondary antibody and DAPI (0.5 micrograms/mL) in blocking buffer for 1 hour at RT.
9. Rinse slides in PBS 3 times, 10 minutes each.
10. Dehydrate with 50%, 80%, 90%, and 95% alcohol.
11. Mount with Vectashield mounting medium.
I have tried this method with many different primary antibodies, all validated for IF. The secondaries are from Vector Laboratories (Fluorescein and Texas-Red conjugated mouse/rabbit anti-IgG). My DAPI staining is not uniform across my slices; there are areas that you can see the nuclei very well, but others on the same slice are very dark). Any help would be appreciated!
What I do as previour answer. I perfuse the animal using 4%PFA and collect the cord followed by post fixation for one day. Immanse in 30% succrose solution for 48 hours and embedded in 4% grlatine followed by OCT compund. For free floating i wash with warm-30-40 degrees 0.01malar PBS and block non specific binding using 10% goat serum for two hours at room temperature. I use triton x 100 dissuolved in pbs while incubating with primary and secondary antibody. The binding i get if fine. However, sometime it depends upon the antibody specificity as well. Could try using high concentration of antibody, increase the incubation time, room temperature/cold room. Hopefully it will help. Thanks
Perfusing in PFA and immersing in sucrose was the method my PI previously used, but this new method was suggested and we are trying to get some results since it is easier. But I may just suggest to my PI that we go back to the original method. Thank you both for your answers!
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