Fluorescent Antibody Dilutions

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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

anti-c-Fos from goat

  • Santa Cruz

anti-rabbit 488 secondary

anti-goat Alexa 555 secondary

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