Hi
I am looking for an optimized protocol for Lc3 and lamp 2 immunofluroscene.currently facing a problem,,have been tried with different conditions as per few papers,but could not able to make correct result.with my protocols I don't see any pattern of immunofluroscence.If any got experience and solution kindly help me by suggestions.
My conditions
Thp1 cell line -differebtiated to macrophage like phenotype
4 % pfa fixed
Permiabilized with triton x or 0.01 % sapo in
Primary ab 3 hrs
Secondary ab 1 hr green channel
Subsequent washes followed
I followed few biblio works saying neat protocols,but I have ended with failure
I would like to receive suggestions that leads good result,I am ready to follow all.kindly share your suggestions or few references ,will be a cool day for me.
I am using macrophage like cell line
Thanks
Sriki
Hi Sriki,
I have an LC3 and LAMP1 protocol that works in primary neurons, microglia and astrocytes. You need to make sure the PFA is fresh for it to work though.
1. Wash cells with 1XPBS twice.
2. Permeablize with 4% PFA in 1XPBS for 10-20 minutes.
3. Block in 2-5% Goat serum(or donkey depending on your fluorescent secondary antibody) in 1XPBS with 0.2%-0.3% TritonX-100 for one hour.
4. Switch to 5% BSA in 1XPBS with 0.2%-0.3% TritonX-100 to dilute your primary antibody. I've had to use 1:200 for LC3 and I use Cell Signal #3868. Keep the primary on over night.
5. Wash in 1XPBS for 30-60 minutes.
6. Switch to secondary antibodies in 2-5% Goat serum in 1XPBS with 0.2%-0.3% TritonX-100 for 60-90 minutes.
7. Wash in 1XPBS for at least 60 minutes.
If you are having trouble with background fluorescence add 0.3M glycine to the buffer containing secondary antibodies.
Good luck,
Monica Holley
Hi Monica,
Thanks for sharing this protocol, I am using LC3antibody polyclonal Rabbit.
I wish to perform this protocol as soon as possible, I will let you know the result soon.
Thanks again
sriki
Hi! To detect Lc3 I don't use triton but I permeabilize with cold methanol (6' at -20°C) after fixation with PFA. Try also leaving overnight at 4°C the primary antibody. The block has to be done with the source serum of your secondary antibody (so if your secondary antibody is produced in goat you need to use goat serum). Moreover I block also with 4% BSA in PBS for 15' and I use 0,1- 1% BSA in PBS to do washings and to resuspend the antibodies.
Hi Sriki,
Hope the following Nature Protocol will help; this is for a super-resolution technique used for Microtubule imaging, but the same principles can be applied here.
Article Direct stochastic optical reconstruction microscopy with sta...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line