Table 1.
Immunoblot and immunofluorescence antibody details
| Protein/Lane, μg |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Antibody (Ab) Target | Primary Ab Supplier | Cortex | Medulla | Ab host | Dilution | Incubation Time | Secondary Ab Supplier | Ab Host and Target | Dilution | Incubation Time |
| Immunoblots | ||||||||||
| AMPK | Cell Signaling | 80 | 37 | Rb | 1:1,500 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h |
| AMPKpT172 | Cell Signaling | 150 | 37 | Rb | 1:1,500 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h |
| Angiotensinogen | Sernia | Sheep | 1:5,000 | O/N | Invitrogen | DAS680 | 1:5,000 | 1 h | ||
| NCC | McDonough | 60 | Rb | 1:5,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | |
| NCCpS71 | Loffing (Zurich) | 30 | Rb | 1:5,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | |
| NCCpS89 | Loffing (Zurich) | 30 | Rb | 1:5,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | |
| NCCpT53 | Loffing (Zurich) | 60 | Rb | 1:5,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h | |
| NHE3 | McDonough | 30 | 40 | Rb | 1:2,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h |
| NHE3pS552 | Santa Cruz | 30 | 40 | Mu | 1:1,000 | 2 h | LiCor | GAM800 | 1:5,000 | 1 h |
| NKAα1 | Kashgarian (Yale) | 2 | 2 | Mu | 1:200 | 2 h | Invitrogen | GAM680 | 1:5,000 | 1 h |
| NKAβ1 | McDonough | 20 | 2 | Rb | 1:500 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h |
| NKCC | C.Lytle (UCR) | 20 | 8 | Mu | 1:6,000 | O/N | Invitrogen | GAM680 | 1:5,000 | 1 h |
| NKCCpT96T101 | Forbush (Yale) | 20 | 8 | Rb | 1:2,000 | 2 h | LiCor | GAR800 | 1:5,000 | 1 h |
| OSR1 | DSTT, Dundee, UK | 80 | 40 | Sheep | 1:1,000 | O/N | Invitrogen | DAS680 | 1:5,000 | 1 h |
| SPAK | Delpire (Vanderbilt) | 20 | 4 | Rb | 1:3,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h |
| SPAKpS373/OSR1pS325 | DSTT, Dundee, UK | 80 | 40 | Sheep | 1:1,000 | 2 h | Invitrogen | DAS680 | 1:5,000 | 1 h |
| αENaC | Loffing (Zurich) | 80 | 37 | Rb | 1:5,000 | O/N | Invitrogen | GAR680 | 1:5,000 | 1 h |
| βENaC | Loffing (Zurich) | 80 | 37 | Rb | 1:15,000 | 2 h | Invitrogen | GAR680 | 1:5,000 | 1 h |
| γENaC | Palmer (Cornell) | 80 | 25 | Rb | 1:2,500 | O/N | Invitrogen | GAR680 | 1:500 | O/N |
| Immunochemistry | ||||||||||
| NCC | McDonough | Rb | 1:5,000 | 1.5 h | Mol. Probes | GAR488 | 1:500 | 1 h | ||
| NCCpT58 | DSTT, Dundee, UK | Sheep | 1:1,600 | 1.5 h | Mol. Probes | DAS568 | 1:500 | 1 h | ||
| NHE3 | McDonough | Rb | 1:100 | 1.5 h | Mol. Probes | GAR488 | 1:500 | 1 h | ||
| NKCC2 | C.Lytle (UCR) | Mu | 1:2,000 | 1.5 h | Mol. Probes | GAM555 | 1:500 | 1 h | ||
| NKCCpT96T101 | Forbush (Yale) | Rb | 1:500 | 1.5 h | Mol. Probes | GAR488 | 1:500 | 1 h | ||
Mu, mouse; Rb, rabbit; O/N, overnight; GAR, goat anti-rabbit; GAM, goat anti-mouse; DAS, donkey anti-sheep.