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Fluorescent Antibody Dilutions (view source)
Revision as of 11:32, 27 August 2013
, 11:32, 27 August 2013created page from 6/13 document
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>
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>
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>
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>
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>
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>
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>
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>
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>
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>
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>
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>