Fluorescent Antibody Dilutions

pCREB dilution Agenda 6/13 Adam Kimbrough Thomas A. Houpt

Materials required

1 rat

Total amount of each reagent required for dilution curves: at least 70 ul to make 7000 ul of 1:100

anti-pCREB from rabbit

• http://www.cellsignal.com/products/9191.html
• $274 per 100uL

anti-c-Fos from goat

• Santa Cruz

anti-rabbit 488 secondary

• http://www.cellsignal.com/products/4412.html
• $138 per 250 uL

anti-goat Alexa 555 secondary

• http://products.invitrogen.com/ivgn/product/A21432?ICID=search-a21432
• $181 per 500 uL

Procedure

Tissue:

• 1 rat inject with 12ml/kg LiCl perfuse at 1h post injection
• Perfuse and postfix for 24h
• 30% sucrose for 48 h
• Cut forebrain to run dilution curve => total of 25 wells

Immuno procedure:

• 30 min Triton 0.2%
• O/N incubation at room temperature with anti-pCREB and anti-c-Fos
• 90 min Secondary incubation with anti-rabbit Alexa 488 and anti-goat Alexa 555
• mount in VectaShield

pCREB dilutions (n=5): 0, 1:500, 1:200, 1:100. 1:100 boiled

anti-rabbit 488 Secondary dilutions (n=5) : 0,1:500, 1:200, 1:100, boiled 1:100

c-Fos dilutions (n=5): 0, 1:500, 1:200, 1:100. 1:100 boiled

anti-goat Alexa 555 Secondary dilutions (n=5) : 0,1:500, 1:200, 1:100, boiled 1:100

Layout of Immuno Wells

Seconday Dilution (1:x)
Primary Dilution (1:x)	∞	500	200	100	boiled 1:100	Total 1°
(uL of 1:100)
∞						0
500						500
200						1250
100						2500
boiled 1:100						2500
Total 2°
(uL of 1:100)	0	500	1250	2500	2500		6750
						6750

Dilution of all antisera:

• make up 1:10 stock
• for 500 uL wells:
• 1:100, add 50 ul 1:10
• 1:200, add 25 ul 1:10
• 1:500 add 10 ul 1:10

Volume of Primary stock (1:100) to add for each anti Fos and anti pCREB:

Seconday Dilution (1:x)
Primary Dilution (1:x)	∞	500	200	100	boiled 1:100	Total 1°
(uL of 1:100)
∞	0	0	0	0	0	0
500	10	10	10	10	10	500
200	25	25	25	25	25	1250
100	50	50	50	50	50	2500
boiled 1:100	50	50	50	50	50	2500
						6750

PBS/BSA to add for Primary:

Seconday Dilution (1:x)
Primary Dilution (1:x)	∞	500	200	100	boiled 1:100	Total 1°
(uL of 1:100)
∞	500	500	500	500	500
500	480	480	480	480	480
200	450	450	450	450	450
100	400	400	400	400	400
boiled 1:100	400	400	400	400	400
							0
						0

Volume of Secondary Stock (1:100) to add for each anti-rabbit and anti-goat:

Seconday Dilution (1:x)
Primary Dilution (1:x)	∞	500	200	100	boiled 1:100
∞	0	10	25	50	50
500	0	10	25	50	50
200	0	10	25	50	50
100	0	10	25	50	50
boiled 1:100	0	10	25	50	50
Total 2°
(uL of 1:100)	0	500	1250	2500	2500		6750

PBS/BSA to add for Secondary:

Seconday Dilution (1:x)
Primary Dilution (1:x)	∞	500	200	100	boiled 1:100
∞	500	480	450	400	400
500	500	480	450	400	400
200	500	480	450	400	400
100	500	480	450	400	400
boiled 1:100	500	480	450	400	400