Hi Paul,

Do you want it fluorescent or transmitted light microscopy?

Hi Irina,

It is for light microscopy.

1. Fixation overnight in 4% paraformaldehyde at 4oC

2. 0.5% NaBH4 in PBS/Triton (0.05 g in 10 ml)…………… ……………..……15’

3. Wash in 0.01 M PBS pH 7.4 ………………………………………….…3x10’ at RT

4. Inhibition of endogenous peroxidase in 3% H2O2 in methanol /PBS at RT, 30-60’

      (20 ml methanol + 75 ml PBS + 4 ml triton + 5 ml 30% H2O2)

5. Wash in 0.01 M PBS pH 7.4 ……………………………………………………6x5’ at RT

6. Pre-incubation in blocking solution    …………………………………………….1h, RT

      (0.5% Triton/PBS 1ml + 1% BSA + 3% NDS (30 mm)

7. Incubation in primary AB in Primary diluent (PD) (at least 18-19 h),

       PD  (0.25 g Gelatin + 1.25 ml triton X-100 + 0.125 Sodium Azide (NaN3) + 250 ml PBS)

                  1. mouse anti-Bassoon diluted 1: ???  (you have to determine which concentration works best for your tissue)

8. Wash in 0.01 M PBS-Triton pH 7.4 ……………………………………………6x5’ at RT

9. Incubation in secondary AB in PBS-Triton ……..…            …………………RT, 4-6h

 Biotinilated (for instance donkey anti-mouse) IgG 1: 500           

      10. Wash in 0.01 M PBS pH 7.4 6x5’ at RT, Make ABC here, 30 minutes in advance!!      

      11. Incubation in ABC complex 1:500 in PBS-Triton 7.4………………………..1-2h, RT

(2ml A + 2ml B + 1 ml PBS-Triton (mixed 30‘ earlier)

12. Wash in 0.01 M PBS pH 7.4 …………………………………………………6x5’ at RT 

13. Development of the reaction with Soft Gray (SG) (1 drop of each component in 2.5 ml PBS) 4-6’

12. Wash in 0.01 M PBS pH 7.4 ……………………………………………….…6x5’ at RT 

13. Wash in AD

14. Dehydration, xylen, Entellan or Glycerin-gelatin to coverslip them

Success!

Irina, thank you so much!

My pleasure, Paul!

Hello again Irina,

I think I have been able to generate positive bassoon staining using your protocol, but I'm not sure....

Would you happen to have an example image of positive bassoon staining? Or could you point me in the direction of a publication that shows it?

Also, would you happen to know much about anatomical distribution of bassoon throughout the mouse brain?

Thanks very much and thank you again for the protocol!

Paul