Hi,
I'm having problems with the Recombinant Anti-MAP2 antibody from Abcam (Recombinant Anti-MAP2 antibody [EPR19691] (ab183830)) antibody on my frozen, 4%PFA perfused, mice brain tissue.
I was wondering if someone could elaborate better the note from the application part of the data sheet; Antigen retrieval: Heated citrate solution (10mM citrate PH 6.0 + 0.05% Tween-20).
To my knowledge AR is not used with frozen tissue, as it is to rough, and can damage the tissue, while also causing tissue detachment from the glass!?
Does someone have any experience with AR and frozen tissue?
I did try using 1/100 dilution, 0,2% triton X-100 and 4%PFA fixation as described in the images part of the datasheet: Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen Mouse Cortex tissue labeling MAP2 with ab183830 at 1/100 dilution.
However I did not observed cytoplasmic staining of my neurons (image as uploaded for reference)!!
My protocol was the following:
4%PFA fixation, 15 min RT
3xPBS, 5min
10 min PBS+0.2% Triton x-100
Blocking solution (3%BSA, Without Triton x-100, 1h)
Anti-MAP2 1:100 dilution in 1%BSA (try both overnight and 2h RT)
3xPBS, 5min
AF-647 1:500, 30-45 min RT
3xPBS, 5 min
DAPI, 5 min
3x PBS, 5 min
dH2O, 5 min
Antifade and coverslip.
The antibody works excellent in IHC-P.
Thanks for the help,
Fran
Your image demonstrates severe cryodamage of the brain tissue, you need to use 15%, 30% sucrose incubation to protect your tissues from ice crystalls formation of the crystals. Antigen retrieval is usually not done and not required for the cryosectioned material. I don't believe your problem is in the lack of antigen retrieval step.
Dear Anton,
I have also noticed that my brain tissue is damaged and was also planning on resolving this problem in the future. However, i'm new in the field and don't have much experience with frozen tissue, so I used the protocol that was giving to me from my lab.
The brains were firstly incubated in 4%PFA for 4h, afterwards 1h in 5% sucrose, 1h in 10% sucrose and finally overnight in 20% sucrose. The next day they were embedded in O.C.T, frozen on dry ice for 30 minutes and stored at -80C.
Back to the antibody problem. I also tested some other antibodies, and they seem to work fine. I was hoping that maybe AR or increasing permeabilization would fix this problem, and that it is not related to the tissue quality.
All the best,
Fran
Your protocol seems fine, you do proper fixation, cryoprotection, and permeabilization. You can try to reduce fixation time or if it is just one antibody that is not cooperating try to change to a different antibody for the same target.
Look into Microwave Cookbook for microscopists of Kok and Boon. Suurmeijer was the scientist who started antigen retrieval, don't have publication at hand, try to find it, gives you a good method. Succes Marani
see also. Marani E et al., Microwave cryostat technique for neuroanatomical studies. J Neurosci methods 22 1987 97-101 and also
Hopwood, D., Coghill, G., Ramsay, J., Milne, G., Kerr, M.
Microwave fixation: Its potential for routine techniques, histochemistry, immunocytochemistry and electron microscopy
(1984) The Histochemical Journal, 16 (11), pp. 1171-1191.
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