Hi Max,

Dearly grateful for your comment! :-))

And,

Please, YES!, If you have a protocol in which you can share it with me, I appreciate it very much.

Your image looks Fantastic, and what an elegant neuron you captured!,

I am going to use the free-floating technique to obtain 30 um sections for confocal imaging.

With the best,

Leila :-)

Hi Leila,

it should work. I usually use the same Neurobiotin (1mg/ml internal solution) and streptavidin 568 (1:500)!

Let me know if you have problems.

Hi to both of you Max and Martina,

And Great thanks for your valuable tips and comments, I appreciate them a lot! :-)

I have looked at a sample (it was developed for clarity technique) yesterday, and notice a high autofluorescence and background staining. It was looking that I stained all other neurons too, which I didn't!

Now, I am going to do the usual IHC technique for 30um thick brain sections, and the only marker I am using is Alexa 568 streptavidin for marking Neurobiotin in the single patched neuron.

I was wondering if you have noticed such an effect (high fluorescence unspecified background) in your samples too?

Again, very grateful for the valuable time which you spent helping me, and the experiences which you shared with me.

With the best,

Leila

Hi again Max,

I have just noticed in the protocol you kindly send me, you are using 1:1000 Alexa568-streptavidin (I am using 1:500 like Martina), only for 60 min and not over the night, right?

I am going to use IHC protocol so:

1. Rinse the sections (30 um) with KPBS 3x10 min.

2. Incubate overnight with KPBS, 1% BSA (Bovine Serum albumin), Triton-X, and Streptavidin conjugated with Alexa 568-streptavidin (1:500), @ +4C.

3. Rinse the sections with KPBS for 3x10 minutes.

4. Mount on pre-treated glass slides with DPX.

Any comments?

Great thanks Max!

I appreciate your valuable time and comments again.

With the best,

Leila :-)