Neuron dead after immuno - (Jan/22/2018 )

Hi,

I am doing Immuno on cultured hippocampal neurons on coverslips. I found that most of my neurons are dead after doing the immuno.

When I use DAPI, I see many neurons, but once I change to fluoroscence, I find most of my neurons dead. Kindly suggest, what can I do to solve this problem?

I am sharing the protocol that I use below:

1) Remove coverslips and leave them at RT for 10 min (on the culture media).

2) Rinse them once with fresh PBS.

3) Fix the coverslips with 500 µL of 4% PFA one ice for 10 min.

4) Wash them 3 times with fresh PBS for 5min each (3 x 5’).

5) Add 1 ml of blocking solution to the coverslip and incubate them in 37ºC for 30 min. In the meantime, prepare the primary and secondary antibody solution.

6) Add 1 ml of primary antibody solution to the coverslips and incubate them at 37ºC for 45 min.

7) Wash the coverslip 3 times with fresh 1X PBS (3 x 5’).

8) Add 1 ml of secondary antibody solution to all coverslips and incubate them at 37°C for 45 min.

9) Remove the antibody solution and wash the coverslip 3 times with fresh PBS and 5 min each in dark (3 x 5’).

10) Leave the coverslips in water in PBS; place a drop of Prolong Diamond at the center of the slide; mount 1 coverslip/slide and place them gently on the prolong Diamond inverted, so that the cells face the mounting media.

11) Leave the slides for 24 h at RT, in dark, for curing the mounting media (coverslip side facing up).

-Mad Researcher-

What do you mean dead? I would expect the neurons to be dead after fixation - this stops all cellular processes.

If you mean that you don't see any staining, then it could be a number of things:

What are you using to permeablize the cells? Most people use something like triton X-100.

Have you titrated the antibody to determine the best concentration for staining?

What is in your blocking solution and antibody solution (salt concentrations, protein %, detergent %)?

Have you tried primary for longer and/or lower temperatures?

Does your primary antibody bind the native form of the protein? Some do, some don't, the product sheet may indicate usefulness in this regard.

Is PFA fixation appropriate for the antibody? You may need to try methanol/acetone fixation protocols or even glutaraldehyde.

What controls do you have?

-bob1-

bob1 on Tue Jan 23 18:04:07 2018 said:

What do you mean dead? I would expect the neurons to be dead after fixation - this stops all cellular processes.

If you mean that you don't see any staining, then it could be a number of things:

What are you using to permeablize the cells? Most people use something like triton X-100.

Have you titrated the antibody to determine the best concentration for staining?

What is in your blocking solution and antibody solution (salt concentrations, protein %, detergent %)?

Have you tried primary for longer and/or lower temperatures?

Does your primary antibody bind the native form of the protein? Some do, some don't, the product sheet may indicate usefulness in this regard.

Is PFA fixation appropriate for the antibody? You may need to try methanol/acetone fixation protocols or even glutaraldehyde.

What controls do you have?

Sorry, I should have been more clear.

I am using Triton-x-100 for permeabilization i.e. I add it to the blocking solution and incubate it at 37°C.

Yes, I did that. But none of them showed any staining with the antibody, but were positive with DAPI.

Blocking solution - 1x PBS + 0.5% Triton-X + Normal goat serum (10%).

I am following a protocol from a paper and doing exactly the same.

It worked only once in December last year, where I got staining in many neurons. The only difference was to my blocking solution, I was adding 3% BSA.

-Mad Researcher-

That looks OK, though triton is a pretty powerful detergent to have for your antibody incubations. I would substitute with IGEPAL-CA630/NP-40 or tween 20.

What happens if you switch back to BSA for the blocking? If you are using goat serum because your antibody is goat - then you have made the wrong choice, you should use a non-related species for blocking. The NGS would be an appropriate as a primary antibody control though.

-bob1-

bob1 on Tue Jan 23 20:32:32 2018 said:

That looks OK, though triton is a pretty powerful detergent to have for your antibody incubations. I would substitute with IGEPAL-CA630/NP-40 or tween 20.

What happens if you switch back to BSA for the blocking? If you are using goat serum because your antibody is goat - then you have made the wrong choice, you should use a non-related species for blocking. The NGS would be an appropriate as a primary antibody control though.

I see most of my neurons are stained if I use BSA for blocking.

My secondary antibody is goat anti mouse (Alexa 488/555). Is it the wrong antibody?

My primary is rabbit. What should I change to?

-Mad Researcher-

You antibodies are fine individually. However, as you have a primary that is rabbit, you need a secondary that is targeting rabbit IgG, not mouse IgG. Your secondary should be goat-anti-rabbit (or something similar).

Having a goat secondary with goat serum is fine, in fact it is the recommended procedure. Just make sure that your primary is never the same species as your block.

-bob1-

bob1 on Tue Jan 23 22:26:32 2018 said:

You antibodies are fine individually. However, as you have a primary that is rabbit, you need a secondary that is targeting rabbit IgG, not mouse IgG. Your secondary should be goat-anti-rabbit (or something similar).

Having a goat secondary with goat serum is fine, in fact it is the recommended procedure. Just make sure that your primary is never the same species as your block.

Sorry, I mis-quoted. I am using Goat-anti-rabbit secondary.

Any suggestions to improve my staining?

One more thing that I remember, I used to store the buffer, blocking solution at RT before, but now I do everything on ice. Do you think that would make a difference?

-Mad Researcher-

It sounds like you need to use the BSA, but you might find it useful to determine if the signal is specific if you can - preincubate some primary antibody with peptide/protein against the epitope(s), and see if this removes the signal.

-bob1-

bob1 on Wed Jan 24 18:07:44 2018 said:

It sounds like you need to use the BSA, but you might find it useful to determine if the signal is specific if you can - preincubate some primary antibody with peptide/protein against the epitope(s), and see if this removes the signal.

Thanks. I am doing another immuno with BSA. I hope it works out.

-Mad Researcher-