Anti-CCSP - Rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Primary lung cells were isolated from 1- to 12-week-old wild-type SPC H2B-GFP or β-actin-DsRed mice, as previously described (10)	Staining with 7-aminoacrinomycin D (Molecular Probes, Eugene, OR) or 4′,6-diamidino-2-phenylindole (Sigma Chemical Co., St. Louis, MO) staining to eliminate dead cells. Cells were fixed with BD Cytofix/Cytoperm (PharMingen), according to the manufacturer’s instructions. Rat IgG2b and rabbit IgG (PharMingen) were used for isotype controls.	Cell sorting was performed with a Cytomation MoFlo (Beckman Coulter, Inc., Brea, CA) or a BD FACS Aria (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).

Upstream tips
Primary lung cells were isolated from 1- to 12-week-old wild-type SPC H2B-GFP or β-actin-DsRed mice, as previously described (10)
Protocol tips
Staining with 7-aminoacrinomycin D (Molecular Probes, Eugene, OR) or 4′,6-diamidino-2-phenylindole (Sigma Chemical Co., St. Louis, MO) staining to eliminate dead cells. Cells were fixed with BD Cytofix/Cytoperm (PharMingen), according to the manufacturer’s instructions. Rat IgG2b and rabbit IgG (PharMingen) were used for isotype controls.
Downstream tips
Cell sorting was performed with a Cytomation MoFlo (Beckman Coulter, Inc., Brea, CA) or a BD FACS Aria (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).

Publication protocol

Primary lung cells were isolated from 1- to 12-week-old wild-type SPC H2B-GFP or β-actin-DsRed mice, as previously described (10), using pan CD45-allophycocyanin (APC), CD31-APC, CD74-fluorescein isothiocyanate (PharMingen, San Diego, CA), and EpCAM-phycoerythrin-cyanine dye (BioLegend, San Diego, CA) with 7-aminoacrinomycin D (Molecular Probes, Eugene, OR) or 4′,6-diamidino-2-phenylindole (Sigma Chemical Co., St. Louis, MO) staining to eliminate dead cells. In the intracellular staining for CD74 (PharMingen), proSP-C (Chemicon, Billerica, MA), and CCSP (Seven Hills Bioreagents, cincinnati, OH), cells were fixed with BD Cytofix/Cytoperm (PharMingen), according to the manufacturer’s instructions. Rat IgG2b and rabbit IgG (PharMingen) were used for isotype controls. Cell sorting was performed with a Cytomation MoFlo (Beckman Coulter, Inc., Brea, CA) or a BD FACS Aria (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).

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