X-gal staining of adult olfactory bulbs

 

Animals perfused in 4% PFA/0.1M sodium phosphate buffer  pH 7.4

 

Post fixed 1 hr in 4%PFA/0.1M sodium phosphate

 

Washed 2 X 15 minutes in Xgal wash (Buffer B)

 

Stained overnight at 37^o in X-gal staining solution(Buffer C)

 

2 X 15 min  X gal wash (Buffer B)

 

Stored at 4⁰ C in 4% PFA

 

Washing Buffer  (Buffer B)

 

 Buffer A

 

    0.1M Phosphate buffer pH 7.4

    2mM MgCl

    5mM EGTA

     Recipe (200 mls)

               197.6 ml 0.1M Phos. buffer

                400 ul 1M MgCl2

                2 mls 0.5M EGTA

 

Buffer B

 

0.1M phosphate buffer, 2mM MgCl , 5mM EGTA(Buffer A)

0.01% NA desoxycholate (AKA deoxycholic acid)

0.02% Nonidet P-400

Recipe (100 mls)

             100 mls fixing buffer

             10 mg Na Desoxycholate  powder

              20 ul NP40

 

Buffer C

     Wash buffer(Buffer B)

     1mg/ml X-gal

     5mM  Kferrocyanide

     5 mM K ferriccyanide

      Recipe (10 mls)

              9.675 mls wash buffer

              125 ul X-gal (80 mg/ml dissolved in dimethyl formamide

              100 ul 500mM potassium ferrocyanide

              100 ul 500 mM potassium ferriccyanide