Hello all,
I am training to stain by Immunofluorescence for Tau protein on mouse cortical neurons, I used Monoclonal mouse Tau antibody( ref: 314011synaptic system) with dilution 1/1000 and also I tried with another Monoclonal mouse Tau antibody (ref:MAB3420Sigma-Aldrich) with dilution 1/1000 as well. For both antibodies I tried with different fixation method, Methanol fixation for 5 mins at-20 ºC/Methanol-EGTA(1mM) for 5 mins at -20ºC/Methanol 5 mins at-20 ºC followed by 4%PFA-4%Sucrose at room temperature and 4%PFA-4%Sucrose at room temperature. My neurons are DIV14 but with these fixation methods the 2 different antibody are not working except with methanol-PFA fixation but I do not think it is specific since I think i should be only positive in axon but I still see the staining every where
So, I wonder if some one have already use these antibodies and manage to have a good staining?
Any recommendation would be appreciated, Thank you in advance
Dear Rayan Dawod ,
I have checked that data sheet for the Sigma Antibody you mentioned in your question. Since the vendor is referring to a concentration of 1 mg/ml in the tube and the staining concentration should be around 5 µg/ml (1:200), you should try higher concentrations.
best wishes
Soenke
Hi! I have some suggestions
Antibody specificity: Make sure the antibodies used are specific for mouse neuron Tau protein. You can check this by looking at product datasheets or by contacting the manufacturer.
Method of fixation: The fixation method can influence strongly the accessibility of the antibody to its target. You’ve tried a number of methods, but maybe you need to try varying the fixative times or temperatures or experimenting with different fixatives like paraformaldehyde or formalin.
Permeabilization: After fixation, cells are normally permeabilized so that antibodies can access their intracellular targets. If you aren’t already doing this, you might want to try adding a step of permeabilization with a detergent such as Triton X-100.
Blocking: A blocking step is usually performed in order to block non-specific binding of the antibody using a protein like bovine serum albumin (BSA) or a serum which matches the host species of the secondary antibody.
Dilution of the antibodies: You will probably have to change the dilution of your primary antibody. Too strong a concentration can lead to non-specific binding whereas too low a concentration might not yield a strong signal.
Controls: Make sure you are using appropriate controls. A negative control where you omit the primary antibody makes it likely that what has been staining specific activity? A positive control result known gives you a way to confirm if your protocol is working correctly.
1. please try to increase your fixation time to 10-12 mins.
2. Use permeabilization step with 0.25%Triton x100 for 10 min.
3. Block with 2-3% BSA made in PBS.
4. Use a dilution of 1:200 for your antibody.
I think this will solve your problem.
Best wishes
Subhajit