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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==
<tab sep=tab>
		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	

</tab>

'''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:'''
<tab sep=tab>
		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	
</tab>

'''PBS/BSA to add for Primary:'''
<tab sep=tab>
		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	
</tab>

'''Volume of Secondary Stock (1:100) to add for each anti-rabbit and anti-goat:'''
<tab sep=tab>
		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
							
</tab>

'''PBS/BSA to add for Secondary:'''
<tab sep=tab>
		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
</tab>