Calf-Intestinal Alkaline Phosphatase Treatment in situ-Killinger 2024
Solji Choi, Bryan Killinger
Abstract
Alpha-synuclein phosphorylated at serine 129 (PSER129) occurs in two pools, non-aggregated (physiological) and aggregated (disease). This protocol allows for the selective dephosphorylation of non-aggregated PSER129 and enhances the specificity and sensitivity immunodetection of aggregated PSER129. Thus, this protocol can be used to differentiate physiological from aggregated PSER129.
Steps
Day 1
Wash free-floating tissue (3 x 10 minutes) in dilution media.
- Dilution media:
| A | B |
|---|---|
| Nacl | 150 mM |
| Tris-HCl pH 7.4 | 50 mM |
| Triton-X100 | 0.5% |
Wash free-floating tissue 0h 10m 0s in dilution media (1/3).
Wash free-floating tissue 0h 10m 0s in dilution media (2/3).
Wash free-floating tissue 0h 10m 0sin dilution media (3/3).
Incubate the samples with 1% Triton X-100 in DM for 0h 10m 0s.
Wash in DM 0h 10m 0s.
Wash the tissues in CIAP buffer (2x10 minutes).
- CIAP buffer: | A | B | | --- | --- | | Nacl | 100 mM | | Tris- Hcl | 50 mM | | Mgcl2, pH 7.9 | 10 mM |
Autoclave and store .
Wash the tissues in CIAP buffer 0h 10m 0s (1/2).
Wash the tissues in CIAP buffer 0h 10m 0s (2/2).
Incubate the tissues with CIAP at a dilution of 1:333 for 24h 0m 0s at 37°Con a shaker.
-
CIAP concentration per bottle: 20 u/μl (Promega, Cat.# M2825).
-
In
500µLCIAP buffer, add1.5µLCIAP (30 units).
Day 2
Wash in DM (2 x 10 minutes).
Wash in DM 0h 10m 0s (1/2).
Wash in DM 0h 10m 0s (2/2).
Heat water bath to 80°C-85°C 1h 30m 0s before the antigen retrieval step.
Place the dish containing sodium citrate buffer in the water bath and heat it for 0h 10m 0s.
- Sodium Citrate Buffer, pH 6.0 (1L):
2.94gSodium citrate-Trisodium salt (Dihydrate) in1000mLDI water. pH 6.0. Add0.5mLTween-20. Mix well.
Wash the tissues in sodium citrate buffer 0h 10m 0s.
Incubate the tissues in the heated sodium citrate buffer for 0h 30m 0s.
Cool the dish containing tissues to Room temperature (at least 0h 20m 0s).
Wash in DM for 10 min x 2 times.
Wash in DM for 0h 10m 0s (1/2).
Wash in DM for 0h 10m 0s(2/2) .
Tissues are now ready for downstream assays.
