Eclipse a1rsi inverted microscope

Paraffin-embedded tissue sections underwent heat-assisted antigen retrieval with citrate buffer (pH 6.0), followed by blocking with donkey or goat serum and incubation with primary antibodies overnight (Supplemental Table 3): Nkx2.1 (Seven Hills Bioreagents, WRAB-1231), Nkx2.1 (in-house antibody, G237), pro-surfactant protein C (Seven Hills Bioreagents, WRAB-9337), ABCA3 (in-house antibody, GP985), and smooth muscle actin (Santa Cruz Biotechnology, A5228). After that, sections were incubated with Alexa Fluor antibody against the host species of the primary antibody (Invitrogen), followed by DAPI (Invitrogen, dilution 1:2000). Sections were mounted with ProLong Gold (Invitrogen). Stained slides were imaged by confocal microscopy for colocalization of fluorescent antibodies at 20× magnification, 1024 × 1024 pixels resolution, on a Nikon Eclipse A1RSi inverted microscope (Nikon Instruments Inc.).

To identify proliferating microglia, we performed double-labeling immunofluorescence for Iba-1 and Ki-67. Antigen retrieval and blocking were conducted as described above, followed by overnight incubation with goat polyclonal Anti-Iba1 (Abcam, Cambridge, MA, cat. #5076, dilution 1:100) and rat monoclonal anti-Ki67 (LifeSpan Biosciences, Seattle, WA, cat. #LS-C175347, dilution 1:50). The following day, sections were washed and incubated with anti-goat Alexa Fluor 594 (Life technologies, Carlsbad, CA, cat. #A-11058, dilution 1:200) and anti-rat Alexa Fluor 488 (Life technologies, Carlsbad, CA, cat. #A-21470, dilution 1:200) for 2 h at room temperature, followed by washing and incubation with DAPI (Life technologies, Carlsbad, CA, cat. #D1306, dilution 1:2000) for 15 min at room temperature. Sections were washed and mounted with ProLong Gold (Life technologies, Carlsbad, CA, cat. #P36930). Stained slides were imaged on confocal microscopy for co-localization at ×40x magnification with 1024 × 1024 pixel resolution on a Nikon Eclipse A1RSi inverted microscope (Nikon Instruments Inc, Melville, NY).