ChAT Protocol

Rabbit Anti-ChAT (R-aChAT; Chemicon AB 143)

 

Perfuse according to protocol with NO post-fix.  ALSO… 4 hrs in 10% sucrose, 12 hrs in 20% sucrose and 48 hrs in 30% sucrose followed by shock freezing in 2-methylbutane on dry ice for 30 seconds.  Can also use cryo-protected tissue.  All steps are to be performed at room temperature unless otherwise specified.

 

 

1.    3 x 10 minute rinse in PBS (pH 7.3)

2.    Wash tissue for 15 minutes in dilution media solution (DMS)

3.    Incubate for 20 minutes in 0.05 M trizma buffered saline (TBS) + 0.1 M sodium periodate

4.    3 x 10 minute rinses in DMS

5.    Incubate for 60 minutes in blocking solution

6.    Incubate for 64 hrs in primary antibody.  1:2000 R-aChAT in primary antibody solution

7.    (4°C; 5 pm – 9am)

8.    3 × 30 minute rinses in DMS

9.    Incubate for 120 min 1:100 bGAR secondary antibody in secondary antibody solution

10. 3 × 30 minute rinses in DMS

11. Incubate for 120  min in AB solution

12. Add 2 drops of reagent A to 5 mL of DMS, mix well, then add 2 drops of reagent B and mix well. 

13. Solution must be made 45 minutes prior to use.

14.  1 x 10 minute wash in 0.05 M trizma buffer (TB; pH 7.6)      

15.  3 x 10 minute washes in 0.1 M imidazole in TB

16.  5 sec - 2 min incubation in 0.05 % 3,3’-diaminobenzidine (DAB) + 0.016 % H2O2

17.  3 × 10 minute rinses in 0.1 M imidazole in TB

 

 

-Solutions on next page-

 

Solutions:

 

0.1 M PBS (pH 7.4)                      0.05 M TBS (pH 7.3)                     DMS

7.1 g Na2HPO4                            12.1 g trizma base                       250 mL triton X-100

8 g NaCl                                   9 g NaCl                                   475 mL TBS

984.9 mL dH2O                           970 mL dH2O  

 

 

TBS + 0.1 M sodium periodate                    Blocking solution                                    0.05 M trizma buffer (pH 7.6)

0.53 g sodium periodate                            24 mL DMS                                           494 mL dH2O      

24.5 mL TBS                                         0.5 mL normal goat serum                          6.05 g trizma base

                                                        0.5 g bovine serum albumin

 

 

Primary/Secondary Antibody solution

100 mL triton X-100                      OR        150 mL triton X-100         

250 mL NGS                                         375 mL NGS

0.25 g bovine serum albumin                      0.375 g bovine serum albumin

24.4 mL 0.1 M PBS                                  36.6 mL 0.1 M PBS

 

 

0.05 M TB (pH 7.6)                                  0.1 M imidazole in TB                               DAB + H2O2

6.05 g trizma base                                  248.3 mL TB                                         20 mL TBS (pH 7.3)

494 mL dH2O                                         1.7 g imidazole                                       10 mg DAB

                                                                                                                 12 mL 30% H2O2