Antibodies for staining: mouse anti-DLL4 (sc-365429, SCBT) and anti-PECAM (sc-376764, SCBT); rabbit anti-ERG (ab92513, Abcam), anti-AQP5 (orb15125, Biorbyt), anti-APLNR (20341-1-AP, Thermo Fisher Scientific), anti-SPC (ab90716, Abcam), anti-PDGFRA (ab203491, Abcam), anti-JAG1 (ab7771, Abcam); rat anti-PECAM (550274, BD Biosciences), anti-Ki67 (11-5698-82, Thermo Fisher Scientific); goat anti-Carbonic Anhydrase IV (CAR4) (PA5-47312, Thermo Fisher Scientific); and chicken anti-β galactosidase (ab9361, Abcam). Antibodies for Western blot: mouse anti-DLL4 (sc-365429, SCBT), anti-HES1 (sc-166410, SCBT), anti-JAG1 (sc-390177, SCBT), anti-PECAM (sc-376764, SCBT), anti-NR2F2 (sc-271940, SCBT); rabbit anti-NICD (4147, Cell Signaling), anti-PDGFRA (ab203491, Abcam), anti-SPC (ab90716, Abcam), and anti-VEGFA (ab46154, Abcam); goat anti-DLL4 (ab7280, Abcam); anti-mouse β-actin (ACTB) (A1978, Sigma). The primers were purchased commercially from Sigma.
Ab90716
Manufactured by Abcam
Sourced in United Kingdom, China
Ab90716 is a lab equipment product. It has a core function, but no further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (4 mentions)
Ab92320, by Abcam (3 mentions)
Ab40873, by Abcam (2 mentions)
Ab106926, by Abcam (2 mentions)
Ab181153, by Abcam (2 mentions)
Alexa fluor 488 igg 53 5381 80, by Thermo Fisher Scientific (2 mentions)
Igg h l rhodamine red conjugate r 6394, by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Lps from escherichia coli o111 b4 l2630, by Merck Group (1 mentions)
Phospho iκbα ser32 14d4, by Cell Signaling Technology (1 mentions)
Anti cd45 30 f11, by Thermo Fisher Scientific (1 mentions)
Anti nf κb p65 sc 109, by Santa Cruz Biotechnology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab52627, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Antibody diluent, by Zytomed Systems (1 mentions)
Clone d2 40, by Agilent Technologies (1 mentions)
25 protocols using ab90716
1
Multiparametric Immunostaining of Lung Tissue
2
Immunofluorescence Staining Protocol
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LPS from Escherichia coli O111:B4 (L2630, Sigma-Aldrich, St. Louis, MO, USA), IKK inhibitor BMS-345541 (401,480, Calbiochem, Darmstadt, Germany), PKD inhibitor CID755676 (476,495, Calbiochem) and proteasome inhibitor MG-132(Calbiochem) were commercially obtained. Primary antibodies against the following proteins were purchased from Cell Signaling Technology (Denver, MA, USA): β-actin (#4967), phospho-IκBα (Ser32) (14D4) (#2859), IκBα (#9242), phospho-PKD (Ser916) (#2051) and PKD (#2052). Other antibodies used were anti-CD45 (30-F11, eBioscience, San Diego, CA, USA), anti-CD32 (93, eBioscience), anti-Cxcl5 (PAA860Mu01, Cloud-Clone Corp, Katy, TX), anti-pan-cytokeratin (C-11, ab7753, Abcam, Cambridge, UK), anti-prosurfactant protein C (pro-SPC) (ab90716, Abcam), anti-podoplanin (bs-1048R, Bioss, Boston, MA, USA) and anti-NF-κB p65 (sc-109, Santa Cruz, Dallas, TX, USA). In addition, CF dye-labeled secondary antibodies, CF488A (20015) and CF555 (20231), were purchased from Biotium (Hayward, CA, USA).
3
Immunofluorescence Assay for Cell Authentication and Notch-1 Translocation
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For the authentication of RLE‐6TN and A549 cells and the detection of Notch‐1 nuclear translocation, IF staining was carried out using anti‐proSPC (ab90716, Abcam) and anti‐Notch1 (ab52627; Abcam), respectively, following the methods described in our previous study.16 DAPI (Beyotime, China) was used to stain nuclei before capturing images. The images were acquired using a fluorescence microscope (Nikon, Japan). The green fluorescence indicated proSPC or Notch‐1 protein, respectively, and the blue fluorescence indicated nuclei.
4
Immunocytochemistry Profiling of Cell Blocks
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Immunocytochemistry from cell blocks was conducted as described elsewhere (Marwitz et al [18 (link)]. 2011) using mouse anti-E-Cadherin (1/400, Clone ECH6, Zytomed Systems, Germany), mouse anti TTF1 (1/100, Clone SPT24, Zytomed Systems, Germany), mouse anti-Podoplanin (1/100, Clone D2–40, Agilent Dako, Santa Clara, USA), rabbit anti-pro-SPC (1/100, polyclonal, ab90716, abcam, Cambridge, UK), mouse anti-Vimentin (1/1000, Clone V6, Zytomed Systems, Berlin, Germany) and polyclonal rabbit anti-Collagen I (1/1000, Abcam, Oxford, UK) diluted in antibody diluent (Zytomed Systems, Berlin, Germany). Negative controls were included under omission of primary antibody in every staining series.
5
Immunostaining of Embryonic Lung Sections
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Lungs from E17.5 embryos were fixed in formaldehyde solution 4% (4078–9001, Klinipath) at room temperature for 2 hours, washed in phosphate buffered saline (PBS) for 1 hour and later embedded in paraffin blocks. 4 μm-thick whole lung sections were processed following the standard deparaffinization and immunostaining protocol. Primary antibodies used were as follows: rabbit anti-proSPC (1:900, Abcam Cat# ab90716, RRID:AB_10674024), and hamster anti-Pdpn (1:1000, Abcam Cat# ab11936, RRID:AB_298718). The following secondary antibodies were used at 1:500: Goat anti-hamster; Alexa Fluor 488 (ab180063, Abcam); Goat anti-rabbit; Alexa Fluor 555 (A-21430, Thermo Fisher Scientific). Nuclei were either stained with Hoechst dye (B2261, Sigma) or DAPI from the Vectashield mounting medium (H1200, VectorLab Inc). Images were captured on a Zeiss LSM 880 (Oberkochen, Germany).
6
Alveolar Epithelial Cell Protein Analysis
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Primary alveolar epithelial cells were isolated and cultivated as described above. SDS-PAGE and Western blot were performed as described earlier [35 ]. The following antibodies were used: Claudin-18 (38–8000, ThermoFisher, 1:500), SP-C (ab90716, Abcam, 1:500), AQ5 (ab92320, Abcam, 1:500), and a-Tubulin (ab89984, Abcam, 1:1000), anti-rabbit-HRP (P0448, DakoCytomation, Glostrup, Denmark, 1:1000), and anti-chicken-HRP (ab97135, Abcam, 1:1000). HRP-activity was visualized with Clarity Western ECL substrate (BioRad, Hercules, USA). The membranes were developed on conventional films in a dark room and analyzed densitometrically after scanning with TINA 2.0 (Raytest GmbH, Germany).
7
Immunofluorescence Staining of Lung Cells
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Briefly, tissues were stained with goat polyclonal IgG to Irp2 (ab106926, Abcam) at a 1:50 dilution or rabbit monoclonal IgG to Irp2 (ab181153, Abcam) at a 1:50 dilution. Co-staining for type II epithelial cells was carried out using a rabbit polyclonal IgG to prosurfactant protein C (Pro-SPC) (ab90716, Abcam) at a 1:50 dilution and for type I epithelial cells using anti-mouse podoplanin alexa Fluor® 488 IgG (53-5381-80, Affymetrix) at a 1:500 dilution. Co-staining for Irp2 and markers of ciliated airway epithelial cells was conducted using a rabbit IgG for acetyl-α-tubulin (Lys40) (D20G3, Cell Signaling) at a 1:50 dilution, and co staining for non-ciliated secretory epithelial cells was conducted using a rabbit polyclonal IgG to uteroglobin (ab40873, Abcam) at a 1:100 dilution. Secondary staining was carried out using goat anti-rabbit IgG (H+L) rhodamine red conjugate (R-6394, Life Technologies) at a 1:500 dilution or donkey anti-goat IgG (H+L) secondary antibody, alexa Fluor® 488 conjugate (A-11055, Life technologies) at a 1:500 dilution. Nuclei were counter-stained using TO-PRO®-3 Iodide (T3605, Life Technologies) at a 1:1000 dilution or Hoechst (33342, ThermoFisher Scientific) at a 1:300 dilution.
8
Immunofluorescence of Airway Epithelial Cells
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Cells on ALI day 7 were fixed with 4% paraformaldehyde, then incubated in the following series of solutions: blocking buffer for 60 min at RT; antibodies against E-cadherin (1:500, ab40772, Abcam), AQP-5 (1:100, ab92320, Abcam), or proSP-C, (1:200, ab90716, Abcam) for 90 min at RT; Alexa 488-conjugated secondary antibody (1:500, ab150077, Abcam) for 60 min at RT; and DAPI solution (1:1000, Dojindo) for 15 min at RT. The cells were rinsed twice with PBS between each step. Images were acquired by a confocal laser scanning microscope (LSM880, Carl Zeiss).
9
Immunofluorescence Analysis of hAT1-like Cells
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Attached hAT1-like cells were fixed with 4% PFA at 4°C for 3 hr. After cells were blocked in PBS with 5% normal donkey serum and 1% Triton X-100 (Sigma-Aldrich), sections were incubated with primary antibodies overnight at 4°C, then washed, and incubated with host matched Alexa Fluor-couple secondary antibodies (1:1000, Jackson ImmunoResearch Laboratories) (1:1000) for 1.5 hr at RT. Following DAPI incubation, slides were mounted. AGER (1:400, R&D SYSTEMS, AF1145), Aquaporin5 (1:200, abcam, ab92320) pro-SFTPC (1:400, abcam, ab90716) were used.
10
Immunohistochemical Analysis of OPN and Surfactant Protein C in Lung Tissue
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Cells were fixed using 4% paraformaldehyde and lung tissues were fixed and embedded with paraffin, and incubated with antibodies against OPN (ab214050; Abcam) and Prosurfactant Protein C (ab90716; Abcam), followed by staining with fluorescein‐linked anti‐rabbit antibody (A0516; Beyotime). The cell nuclei were stained with DAPI.
The expression of OPN proteins was detected and measured by immunohistology. Sections were incubated with the primary antibody (ab214050; Abcam) at 4°C overnight and covered with the corresponding secondary antibody (ab205718; Abcam). Sections were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. The ratio of integral optical density (IOD) of OPN in the area was Mean Density.21 Images were analyzed with Aipathwell.
The expression of OPN proteins was detected and measured by immunohistology. Sections were incubated with the primary antibody (ab214050; Abcam) at 4°C overnight and covered with the corresponding secondary antibody (ab205718; Abcam). Sections were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. The ratio of integral optical density (IOD) of OPN in the area was Mean Density.
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