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
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 |