Neuron dead

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Neuron dead

Postby newbie17425 » Jan 23 2018 12:44 am

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).
newbie17425
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Re: Neuron dead

Postby r.rosati » Jan 23 2018 7:25 pm

Hi,
by "dead" you mean no fluorescence detected, correct?
The reasons might vary. Have you tried different dilutions of your antibodies? Do you have any sort of positive control to troubleshoot the steps, i.e. another primary that surely works; another secondary that works; another cell type that is positive by this protocol?
Also, would you say that it's a case of most neurons unstained, or could it be a case of varying background with no actual specific staining?
Best,
Roberto
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Re: Neuron dead

Postby newbie17425 » Jan 23 2018 8:03 pm

r.rosati wrote:Hi,
by "dead" you mean no fluorescence detected, correct?
The reasons might vary. Have you tried different dilutions of your antibodies? Do you have any sort of positive control to troubleshoot the steps, i.e. another primary that surely works; another secondary that works; another cell type that is positive by this protocol?
Also, would you say that it's a case of most neurons unstained, or could it be a case of varying background with no actual specific staining?
Best,
Roberto


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. Also, I used Alexa 488 instead of 555. Does that make a huge difference?
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Posts: 138
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