Af647 donkey anti rabbit

Mouse kidney tissues were fixed in 4% formalin for 2 h at RT and frozen in OCT after dehydration in 304%4 sucrose overnight. Using 5–10 μm cryosections, slides were blocked in 5% donkey serum followed by 1-h incubation of the primary antibody, washing three times for 5 min in PBS, and subsequent incubation of the secondary antibodies for 45 min. Following DAPI (4′,6-diamidino-2-phenylindole) staining (Roche, 1:10,000) the slides were mounted with ProLong Gold (Invitrogen, #P10144). Cells were fixed with 3% paraformaldehyde followed by permeabilization with 0.3% TritonX. Cells were incubated with primary antibodies and secondary antibodies diluted in 2% bovine serum albumin in PBS for 60 or 30 min, respectively. The following antibodies were used: anti-Runx1 (HPA004176, 1:100, Sigma-Aldrich), AF647 donkey anti-rabbit (1:200, Jackson Immuno Research).

Extracted upper parts of the hearts were fixed for 2 h in 4% paraformaldehyde (Carl Roth) at RT. These parts were then transferred to 30% sucrose solution in PBS and kept at 4 °C overnight to prevent ice crystal formation. Samples were then embedded in Tissue-Tek O.C.T. Compound (Sakura) and quickly frozen and stored at −80 °C until sectioning. In total, 4 μm cryosections were then washed in PBS and permeabilized in PBST-0.1% (PBS with 0.1% Triton X-100 (Sigma-Aldrich). Blocking was performed in 10% BSA in PBS, followed by 1 h incubation of primary antibody. Slides were washed three times for 5 min in PBS and incubated afterwards with secondary antibody for 30 min. Lastly, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) staining (Roche, 1:10,000) and slides were mounted with ProLong Gold (Invitrogen, P10144). Used antibodies are listed here: Anti-Mouse CD31 (BD Biosciences, 1:100, 553370), Thrombospondin-4 Antibody (Novus Biologicals, 893655, 1:100) and Ki-67 Monoclonal Antibody (SolA15) (eBioscience, 14-5698-80, 1:100). AF488 donkey anti goat (Jackson Immuno Research, 1:200), and AF647 donkey anti-rabbit (Jackson Immuno Research, 1:200) and AF647 donkey anti-rat (Jackson Immuno Research, 1:200).

The celiac ganglia were dissected from MeAàVMH^hM3Dq or mCherry control mice euthanized 2h after CNO administration. The tissue was post-fixed in 4% paraformaldehyde at 4°C overnight, cryo-protected in 30% sucrose (Sigma-Aldrich, 50389) in PBS, embedded in O.C.T Compound (Thermofisher Scientific, Watham, MA; 23–730-572), frozen at −80°C, and sectioned at 10μm thickness. Slides were washed in 0.03% PBT (3 × 5 minutes), incubated in blocking solution overnight at 4°C (2% normal donkey serum, 3% bovine serum albumin in 0.03% PBT), incubated in primary antibodies for 48h at 4°C (Cell Signaling anti-cfos – 1:100; abcam chicken polyclonal to tyrosine hydroxylase [#ab76442] – 1:500), washed in 0.03% PBT (3 × 5 minutes), incubated in secondary antibodies for 2h at RT (Jackson AF-647 donkey anti-rabbit – 1:250; Jackson AF-594 donkey anti-chicken – 1:500), and washed in 0.03% PBT (3 × 5 minutes). After staining, tissue sections were mounted with DAPI counterstain (Fluoromount).