Fig 1: Immunocytochemistry of (A.) type II cells or (B.) alveolar macrophages isolated from wild type and NPC mutant mice.A. Type II cells were isolated from NPC1 (Top) or NPC2 (Bottom) mutant mice or their corresponding wild type littermates and placed in culture for 24 hrs. The cells were fixed and stained with anti-ABCA3 antibody (green) to mark the lamellar body limiting membrane or filipin (Fil, gray or blue) to mark cholesterol. The merged pictures with anti-ABCA3 in green and filipin in blue are enlarged. Scale bar = 5 µm. B. Alveolar macrophages from NPC mutant mice contain cholesterol. Alveolar macrophages were isolated from the lung lavage from NPC1 (Top) or NPC2 (Bottom) mutant mice or their corresponding wild type littermates and placed in culture for 2 hrs. The cells were fixed and stained with filipin which labels free unesterified cholesterol. Lt, Phase micrograph; Fil, Filipin stain in gray. Merge of phase and filipin (blue) are enlarged. Scale bar = 10 µm.
Fig 2: Light micrograph of mutant mice lungs stained with hematoxylin and eosin.Lungs from BALB/c wild type (95 days), NPC1 mutant (70 days) and NPC2 mutant mice (88 days). NPC1 and NPC2 mutant mice show “nests” of macrophages and alveolar macrophages with large inclusions.
Fig 3: Size of mouse lamellar bodies.Using electron microscopic photographs of type II cells from wild type, NPC1 and NPC2 mutant mice, the size of the lamellar bodies was analyzed using ImageJ. Top. Electron micrographs of typical type II cells from wild type (WT, left), NPC1 (middle) and NPC2 (right) mutant mice. LB, lamellar body. Bottom. Histogram of the lamellar bodies from wild type (white triangles), NPC1 (gray circles) and NPC2 (black circles) type II cells. Frequency of each lamellar body size in micron2 is expressed as a % of the total numbers of lamellar bodies. Inset: size of lamellar bodies in grouped bins. Wild type, 60 type II cells, 451 LBs, 3 mice; NPC1, 53 type II cells, 435 LBs, 3 mice; NPC2, 37 type II cells, 459 LBs, 3 mice. *Statistically significant difference, P<0.05. E. Lamellar body sizes in grouped bins of ranges of areas.
Fig 4: NPC proteins in mouse lungs.Western blot of wild type (W) littermates, NPC1 (Mut) or NPC2 (Mut) mutant mouse lungs using anti-NPC1 or -NPC2 antibody. β-actin used as a loading control. 30 µg protein/lane.
Fig 5: Surfactant protein-A (SP-A) content of (A) lungs and (B) surfactant isolated from wild type (W) or NPC mutant (NPC1, NPC2) mice.Left, Western blots of SP-A or actin. Right, quantitation of Western blots. A. Arbitrary units (AU) of SP-A relative to actin from lungs of NPC1 or NPC2 mutant mice (C1 or C2) or age-matched wild type controls (W1 or W2) (n = 6–8) or B. Arbitrary units of SP-A in surfactant (n = 4–9). All samples were loaded at equal protein values. *Significant difference from wild type, (P<0.05). The two SP-A bands are due to differences in glycosylation.
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