960 ml HPLC grade water

40 ml HPLC grade methanol

7.4 g citric acid anhydrous

7.4 g sodium acetate anhydrous

0.16 g EDTA (Ethylenediaminetetraacetic acid disodium salt dehydrate)

0.08 g OSA (1-Octanesulfonic Acid Sodium Salt)

[use HPLC grade O-phosphoric acid (85%) or 6.25 N sodium hydroxide to pH the mobile phase; Fisher Cat # A260-500]

Catalog numbers from suppliers:

HPLC Water, Fisher

HPLC Methanol, Fisher Cat # A452-4

Citric acid anhydrous, Fluka Cat # 27487

Sodium acetate anhydrous, Fisher Cat # S209-500

Ethylenediaminetetraacetic acid disodium salt dehydrate, Sigma Cat # ED2SS

1-Octanesulfonic Acid Sodium Salt, Sigma Cat # O-0133

O-phosphoric acid (85%), Fisher Cat # A260-500

6.25 N sodium hydroxide, Fisher Cat # SS263-500

12.06 g of sodium phosphate dibasic

50.25 mg (0.05025 g) of EDTA (Ethylenediaminetetraacetic acid disodium salt dehydrate)

150 mL methanol

in 850 mL HPLC water

pH 6.50

[use HPLC grade O-phosphoric acid (85%) or 6.25 N sodium hydroxide to pH the mobile phase; Fisher Cat # A260-500]

Catalog numbers from suppliers:

HPLC Water, Fisher

HPLC Methanol, Fisher Cat # A452-4

Ethylenediaminetetraacetic acid disodium salt dehydrate, Sigma Cat # ED2SS

O-phosphoric acid (85%), Fisher Cat # A260-500

6.25 N sodium hydroxide, Fisher Cat # SS263-500

Sodium phosphate dibasic, Fisher Cat # S374-3

100 ml DI water

300 mg gelatin

1. 300 mg gelatin, pour into a 250 ml Erlenmeyer flask.
2. Add 100 ml DI water to flask
3. Microwave on high for 40-45 sec (careful that it does not boil over; if gelatin does not dissolve into solution, increase microwave time).
4. Non-microwave option: stir solution with stir bar, while heating solution, until it gelatin dissolves into solution (again, be careful to not let it boil over). Step 4 is more time consuming than step 3.

Catalog numbers from suppliers:

Gelatin, Fisher Cat # G8-500

MATERIALS	8%	10%	12.5%	15%	17.5%
29:1 Acrylamide	2.2 mL	2.7	3.3	4.0	4.67
4X Lower Tris	2.0 mL	2	2	2	2
ddH2O	3.8 mL	3.25	2.65	1.95	1.3
APS	40 uL	40 uL	40 uL	40 uL	40 uL
TEMED	5 uL	5 uL	5 uL	5 uL	5 uL
Total Vol:	8 mL	8 mL	8 mL	8 mL	8 mL

Separation gel:

Stacking gel:

MATERIALS
29:1 Acrylamide	0.7 mL
4X Upper Tris	1 mL
ddH2O	2.3 mL
APS	20 uL
TEMED	2.5 uL
Total Vol	4 mL

Note: These recipes are for single gel, for double gels simply double everything.

350 ml 0.1 M PB solution (pH 7.35)

150 ml ethylene glycol

100 μg sodium azide

150 g sucrose

Final cryoprotectant concentration will be: 30% ethylene glycol, 30% sucrose, 0.00002% sodium azide, in 0.1 M PB (pH 7.35).  Prepare this solution under the hood.

1. Pour 250 ml of 0.1 M PB into 1 L flask.
2. Pour 150 ml of ethylene glycol into the flask.
3. Add 100 μg sodium azide.
4. Stir for 30 min
5. Add 150 g of sucrose
6. Stir for 2 hr
7. Bring volume up to 500 ml with 0.1 M PB
8. Store cryoprotectant solution in refrigerator (+4ºC)

Cresyl violet staining for mounted slides.

1. 95% ethanol          ……………………………   10 min
2. 70% ethanol          ……………………………   1 min
3. 50% ethanol          ……………………………   1 min
4. dH2O                      ……………………………   2 min
5. dH2O                      ……………………………   1 min
6. Cresyl violet stain*…………………………… 10 min
7. dH2O                      ……………………………   2 min
8. 50% ethanol          ……………………………   1 min
9. 70% acid ethanol+ …………………………   2 min
10. 95% ethanol       ……………………………   2 min
11. 95% ethanol       ……………………………   30 seconds
12. 100% ethanol     ……………………………   1 min
13. Xylene                  ……………………………   3 min
14. Air dry and coverslip.

* Cresyl violet stain: add 1.25 g cresyl violet acetate and 0.75 mL glacial acetic acid to 250 mL of warm dH2O, then cool and filter.

+ 70% acid ethanol: 2 mL glacial acetic acid in 200 mL 70% ethanol.

Neuroendocrinology and Motivation Lab

NE : Norepinephrine (Arterenol)

DOPAC : 3, 4-dihydroxyphenylacetic acid

DOPAMINE: 3-hydroxytyramine

5-HIAA : 5-hydroxy-3-indoleacetic acid

HVA : Homovanilic acid (4-hydroxy-3-methoxyphenylacetic acid)

5-HT : 5-hydroxytryptamine (serotonin)

For 1 mg/ml concentration of individual STOCK A solutions:

NE                   9.45 mg/ 5 ml of HPLC grade water

DOPAC          5.00 mg/ 5 ml              “

DA                  5.00 mg/ 4.03 ml         “

5-HIAA          5.00 mg/ 5 ml              “

HVA               5.00 mg/ 5 ml              “

5-HT               5.00 mg/ 2.17 ml         “

5-HTOL          5.00 mg/ 5 ml              “

For 1 μg/ml concentration of individual STOCK B solutions:

5 μl of STOCK A/ 4995 μl of HPLC-grade water

For working solution:

80 μl of STOCK B/ 4920 μl of HPLC grade water to get a 16 pg/μl concentration

40 μl of STOCK B/ 4960 μl of HPLC grade water to get an 8 pg/μl concentration

20 μl of STOCK B/ 4980 μl of HPLC grade water to get a 4 pg/μl concentration

 

And so on…