Anti-Prosurfactant Protein C antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Prosurfactant Protein C antibody (AB90716)

ab90716 staining Prosurfactant Protein C in A549 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab90716 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Prosurfactant Protein C antibody (AB90716)

IHC image of Prosurfactant Protein C staining in a section of formalin-fixed paraffin-embedded normal human lung performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab90716, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Prosurfactant Protein C antibody (AB90716)

Immunohistochemical analysis of formalin fixed paraffin embedded human lung labelling prosurfactant rotein C with ab90716 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab90716 Anti-Prosurfactant Protein C antibody was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IP

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Immunoprecipitation - Anti-Prosurfactant Protein C antibody (AB90716)

Prosurfactant Protein C was immunoprecipitated using 0.5mg Mouse Lung tissue, 5μg of Rabbit polyclonal to Prosurfactant Protein C and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Mouse Lung tissue lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab90716.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 23kDa; Prosurfactant Protein C

All lanes:

Immunoprecipitation - Anti-Prosurfactant Protein C antibody (ab90716)

Predicted band size: 21 kDa

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Exposure time: 20min

• WB

Lab

Western blot - Anti-Prosurfactant Protein C antibody (AB90716)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602).

In Western blot, Anti-6X His tag® antibody [AD1.1.10] - Loading Control (ab15149).

All lanes:

Western blot - Anti-Prosurfactant Protein C antibody (ab90716) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg

Lane 2:

293T transfected with a human SFTPC expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 3:

293T transfected with a mouse SFTPC expression vector containing a His-tag, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 21 kDa

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Exposure time: 4s

• WB

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Western blot - Anti-Prosurfactant Protein C antibody (AB90716)

All lanes:

Western blot - Anti-Prosurfactant Protein C antibody (ab90716) at 1 µg/mL

Lane 1:

Lung (Human) Tissue Lysate at 10 µg

Lane 2:

Lung (Mouse) Tissue Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 21 kDa

Observed band size: 25 kDa,28 kDa,90 kDa

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Exposure time: 3min

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Prosurfactant Protein C antibody (AB90716)

Flow cytometry (intracellular) analysis of transfected 293T cells. Cells were fixed with 4% PFA for 10 minutes and permeabilised with 90% methanol before staining for SFTPC in 293T cells transfected with a mouse SFTPC expression vector containing a myc-His-tag® using antibody ab90716 at a 1/1000 dilution (1 μg/ml) (middle panel). Staining was compared with 293T cells transfected with an empty expression vector containing a myc‑His‑tag® (right panel) and with an isotype control, rabbit monoclonal IgG (ab172730) (left panel). Goat anti‑rabbit IgG (Alexa Fluor® 488, ab150081) was used as the secondary antibody at a 1/5000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Prosurfactant Protein C antibody (AB90716)

Flow cytometry (intracellular) analysis of transfected 293T cells. Cells were fixed with 4% PFA for 10 minutes and permeabilised with 90% methanol before staining for SFTPC in 293T cells transfected with a human SFTPC expression vector containing a myc-His-tag® using antibody ab90716 at a 1/1000 dilution (1 μg/ml) (middle panel). Staining was compared with 293T cells transfected with an empty expression vector containing a myc‑His‑tag® (right panel) and with an isotype control, rabbit monoclonal IgG (ab172730) (left panel). Goat anti‑rabbit IgG (Alexa Fluor® 488, ab150081) was used as the secondary antibody at a 1/5000 dilution.

• WB

Lab

Western blot - Anti-Prosurfactant Protein C antibody (AB90716)

All lanes:

Western blot - Anti-Prosurfactant Protein C antibody (ab90716) at 1 µg/mL

All lanes:

Lung (Human) Tissue Lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Predicted band size: 21 kDa

Observed band size: 25 kDa,28 kDa,45 kDa

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Exposure time: 2min

• IHC

CiteAb

Immunohistochemistry - Anti-Prosurfactant Protein C antibody (AB90716)

Immunohistochemistry-immunofluorescence using Anti-Prosurfactant Protein C antibody, ab90716. Publication image from Adler, K. B. et al., 2020, Nat Commun, 32111836. Legend direct from paper.

Lung repair and fibrosis in mice after silica-injury.a The experimental study schematic of the silica-induced fibrosis study in A/J mice; n = 10 biological independent animals per group. b Representative TUNEL staining of apoptotic cells for each treatment group and quantification of percent of Tunel positive cells; Scale bar = 50 µm; each dot represents data from one animal; n = 5 biological independent animals. c Representative H&E staining. Scale bar = 100 µm. d Quantification of fibrosis by Ashcroft score; each dot represents data from one animal; n = 8 biological independent animals. Ashcroft score was performed by averaging the score from two blinded and one non-blinded scorer. e Gomori’s trichrome staining : muscle fibers (red), collagen (blue), nuclei (black-purple) and erythrocytes (red). Scale bar = 100 µm. f Representative picrosirius red staining; Collagen types I and III (red). Scale bar = 100 µm. g Quantification of pulmonary hydroxyproline levels; each dot represents data from one animal; n = 4 biological independent animals. h Representative immunostaining of lung sections for aquaporin 5 (AQP5), pro-surfactant protein C (Pro-SPC) and von Willebrand Factor (vWF) Scale bar = 50 µm. i–k Quantification of percent pixel intensity of AQP5+ (i), ProSPC+ (j), and vWF+ (k). Each dot represents data from one animal; n = 4 biological independent animals. Throughout, data are mean ± s.d. P-value as indicated by non-parametric one-way ANOVA; NS, not significant.

• WB

CiteAb

Western blot - Anti-Prosurfactant Protein C antibody (AB90716)

Western Blotting using Anti-Prosurfactant Protein C antibody, ab90716. Publication image from Adler, K. B. et al., 2020, Nat Commun, 32111836. Legend direct from paper.

LSC-Sec inhalation treatment promotes alveolar repair.a Representative immunostaining of lung sections for von Willebrand Factor (vWF), pro-surfactant protein C (Pro-SPC) and aquaporin 5 (AQP5). Scale bar = 100 µm. b–d Quantification of percent pixel intensity of vWF+ (b), percent ProSPC+ nuclei (c), and percent pixel intensity of AQP5+ (d); each dot represents data from one animal; n = 12 biological independent animals. e–f Immunoblot analysis of aquaporin 5 (AQP5), pro-surfactant protein C (Pro-SPC), von Willebrand Factor (vWF), alpha smooth muscle actin (αSMA), SMAD3, matrix metalloproteinase 2 (MMP-2), and beta-actin loading control (B-actin) from lung protein lysate (e) with corresponding quantification of protein levels as fold of sham control (f); each dot represents data from one animal; n = 3 biological independent animals. g–h Representative cytokine array with quantification of relative intensity (g) and corresponding quantification of relative intensity (h). Throughout, data are mean ± s.d. P-value as indicated by non-parametric one-way ANOVA.

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