Goat anti sp c

Whole mount X-gal stained brown adipose tissue and lung tissue from P5 mice was embedded in paraffin, and sectioned in 8 µm thick sections. Sections were deparaffinized through graded series of EtOH, and submitted to heat-induced antigen retrieval in 0.01M-citrate buffer or in Target Retrieval Solution, citrate pH 6.0 (Dako Cytomation, S2369). Blocking was in PBS/1% bovine serum albumin/0.5% triton X-100 for 30–60 min at room temperature. Incubation with primary antibodies was in PBS/0.5% BSA/0.25% triton X-100 at +4°C over night: rabbit-anti-beta-galactosidase (1∶50) (MP Biochemicals/Capell 55976); goat anti-SPC (1∶50) (Santa Cruz, sc7706). Mouse-anti-human ASMA (alpha-smooth muscle actin) conjugated to Cy3 (1∶100) (Sigma C6198) was incubated together with the secondary antibodies. Sections were washed in PBS and incubated with secondary antibodies in PBS/0.5% BSA/0.25% triton X-100 for 30–60 min at room temperature: goat-anti-rabbit Alexa Fluor-488 (1∶200) (Life Technologies); donkey-anti-goat Alexa Fluor-488 (1∶200) (Molecular Probes); donkey-anti-rabbit Alexa Fluor-568 (1∶200) (Molecular Probes). Washed sections were mounted with ProLong Gold anti-fade reagent (Invitrogen). Imaging was done in an SP8 confocal microscope (Leica). X-gal staining was visualized with transmitted light.

Cryo sections (14–20 μm) of Pdgfra^GFP lungs were washed in PBS to remove OCT and blocked in 1% bovine serum albumin + 0.5% triton X‐100 in PBS overnight at 4°C. Primary antibodies diluted in 0.5% BSA + 0.25% Triton X‐100 solution were incubated overnight at 4°C. Sections were washed in PBS and secondary antibodies were incubated in 0.5% BSA + 0.25% Triton X‐100 solution for 1 h at room temperature. After washing, samples were mounted in ProLong Gold Anti‐fade reagent with DAPI (Invitrogen, P36931). Primary antibodies: rabbit anti‐E‐Cadherin (1:200) (CST, 3195S), mouse‐anti‐human Asma (alpha‐smooth muscle actin) conjugated to Cy3 (1:200) (Sigma‐Aldrich, C6198), goat anti‐SPC (1:50) (Santa Cruz, sc7706). Secondary antibodies: donkey‐anti‐goat Alexa Fluor‐568 (1:200) (Molecular Probes); donkey‐anti‐rabbit Alexa Fluor‐647 (1:200) (Molecular Probes).