Hi Jingliang,

I don't totally understand the protocol you used.

If your primary antibody was direclty conjugated, it might be interesting for you to actually use a secondary antibody that is conjugated with a  fluorophore, that should increase your signal. However, there would still be the question of the speficity of your staining.

For that, you should also image a section that was stained with only the secondary antibody to assess the background staining. This shoudl tell you if your staining is specific.

If your signal is really low despite the use of a secondary, there is kits to enhance the signal, for that, you should go have a look to the TSA amplification kits

Good luck!

Hi Jingliang,

I don't totally understand the protocol you used.

If your primary antibody was direclty conjugated, it might be interesting for you to actually use a secondary antibody that is conjugated with a  fluorophore, that should increase your signal. However, there would still be the question of the speficity of your staining.

For that, you should also image a section that was stained with only the secondary antibody to assess the background staining. This shoudl tell you if your staining is specific.

If your signal is really low despite the use of a secondary, there is kits to enhance the signal, for that, you should go have a look to the TSA amplification kits

Good luck!

I second Thibault's response, the primary delete will address specificity, once that is confirmed you can then proceed to optimizing your assay.

 Hi  Thibault,

Thank you for your response.

My protocol is shown as follow

1, Dry up the fresh frozen sections with 37 degree incubator.

2, Treated by acetone for 20 min.

3, Block sections with normal goat serum for 30 min, room temperature.

4, Incubate with APC conjugated primary antibody for 3h, room temperature.

5, Incubate with DAPI for 10 min, room temperature.

Actually, I did not use secondary antibody. I used to stain another kind of tissue sections with  only APC conjugated primary antibody and I could get much stronger signal in these sections.

Will secondary antibody combine with primary antibody which is conjugated with fluorophore? 

Regards, 

Hi Jeffrey,

Thank you so much for your reply.

In fact, I did not use secondary antibody. 

Will secondary antibody combine with the primary antibody which is conjugated with fluorophore?

Regards.

It's very possible that the 2nd ab will still bind to your 1st ab-APC. 2nd ab are usually polyclonal antibodies which bind to many different epitopes on your 1st ab. Unless the conjugation of APC to your 1st ab blocks all the binding sites, which is unlikely.

 Hi Chi-Fu,

Thanks for your response. I will have a try by using a secondary Ab.

Regards.

You might try the following things:

1. Formalin fixation

2. Use avidin biotin system for the 2ndary ab. I also got very low staining with labeled primaries.

3. Try a different primary ab. It is possible that the antigen site is somehow masked or inaccessible in your sample.