I am trying to find the best method to double-immunolabel NMJs in leg muscles of mice. I find alpha-bungarotoxin labels the AchR in the muscle quite well and have used neurofilament to label the nerve component. The results have been okay I suppose with PFA-fixed muscle tissue sectioned at 20 um thickness, but I was wondering if anyone had any experience with this and had some tips for the best co-labeling and imaging results. Some studies combine neurofilament with antibody against SV2. Is this recommended overall or are results similar to neurofilament alone?
Hi,
generally we use a co-staining a-bungarotoxin and synaptophysin (with or without neurofilament both are rabbit polyclonal antibodies): Synaptophysin will mark the synaptic vesicles only. For tissue observation on cryostat sections, muscle should be frozen using precooled-isopentane in liquid nitrogen (better structure conservation).
For whole mount staining on fresh tissue (soleus, tibialis..), you should includebefore fixation a collagenase step for 15 minutes (1mg/ml), wash briefly with PBS1X then fixed the sample with formaldehyde 1% in PBS1 hour, then PBS-glycin 0.1M overnight-Permeabilization with Triton 100X 1% 8 hours depending on the size of the tissue- incubation with your antibodies overnight- wash your samples extensively (3 times PBS 1X for at least one hour)- Incubation with your secondary antibodies overnight- wash PBS-BSA 3% the complete day followed by three washes PBS1X.
It is the long protocol...but it works
Best,
Hi Chandler,
we normally use tuj1 antibody labelling neurons, it makes a very nice staining, see confocal image.
Frederic and Cesare,
Thank you both for the suggestions. Also, Cesare, I appreciate you providing the confocal image of your co-localization using Tuj1 antibody. Very nice innervation of the NMJ. I will keep these suggestions in mind in the next experiment.
Chandler
Chandler and Frederic,
What brand would you suggest for bungarotoxin antibody?
I wish to try it on tibialis anterior by immunofluorescence.
Can both bungarotoxin and neurofilament replace the conventional silver cholinesterase method?
Thank you
Hi Wong,
bungarotoxin is not an antibody is a toxin binding acetylcholine receptor, normally is already conjugated with a fluorophore, molecular probes has a wide choice.
Good luck
Hello Frederic,
I have some questions for you:
1) Could you recommend some reference for the protocol you suggested? Is the collagenase treatment coming from Evans et al., 1976?
2) Also, what type of collagenase?
3) Can you do whole mount IF on tibialis? I thought TA was too thick to do a whole mount. I am trying to stain very thick sections though, like 250 micron; bungarotoxin works beautifully but the pre-synaptic marker (I use VachT) is a bit faint and seems to not reach all synapses (the most superficial I think are those that get the antibody while the deepest ones don't, even though they are bungaro-positive)
4) What is the purpose of PBS/glycin o/n?
Thank you,
Giulio
Hello,
Which neurofilament antibody do you recomend in mouse? I have to buy one to use in combination with synaptophysin and I dont find one that hard convince me. I would be gratefull if you could help me.
Cristina,
I have used chicken anti-NF from Aves Labs with good results in combination with a donkey anti-chicken 488 fluorescent secondary from Jackson Immunoresearch. Here is a link to the primary I used from Aves. I found that I had to vary antibody concentration with different lots, so be sure to test concentrations with each vial you get to make sure you optimize for your labeling.
http://www.aveslab.com/products/neuronal-cell-markers/neuronal-markers/nf-h-neurofilament-200-kda-chicken-polyclonal-antibody/
Binney,
Yes, you can use only alpha-bungarotoxin if you are just interested in labeling AchRs.
Is there any specified protocol for AChR staining using alpha bungarotoxin .Please mention me some reference.
Binney,
When we use it, it is quite simple for muscle AchR labeling. We use Alexa Fluor 555-conjugated α-bungarotoxin (α-BTX) from Molecular Probes/Invitrogen at 1:1000 dilution in PBST and incubate the muscle tissue for about 45 min room temperature. Since the bungarotoxin is fluorophore conjugated, the muscle can be mounted and the AchRs visualized immediately. This is the easiest way. This step can also be carried out with fluorescent secondary antibody labeling of pre-synaptic markers like neurofilament and synaptophysin to save time, as long as you use separate fluorescent labeling for each to distinguish them from each other. If you search "NMJ AND bungarotoxin" in PubMed, you will find various papers with similar, though different protocols on how they applied alpha-bungarotoxin to label AchRs. Hope this helps.
Chandler
Sir i want to study NMJ in gastronemius and soleus muscles.So is it effective to use alpha bungarotoxin staining to label AChR in NMJ in case of these muscles or there is another way to study the NMJ
Binney,
Yes. If you are interested only in looking at the AChRs, you can use alpha-bungarotoxin labeling in these muscles. I use it routinely to label AChRs in gastrocnemius muscle.
Best,
Chandler
May I ask If Alpha- bungarotoxin is in a conjugated form
- Alexa Fluor 488, can I co-label with primary antibody to NF200, followed by secondary antibody to the primary?
The double staining that I did previously involves two primary antibodies, a common serum and two secondary antibodies
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