CFSE-stained T cells (5 × 104 cells per transwell) migrated for 4 h across endothelial monolayers to CCL19 (0.5 μg ml−1), fixed for 20 min at 4 °C with 4% (w/v) paraformaldehyde (Affymetrix), and paraformaldehyde then neutralized for 10 min at 4 °C with PBS 0.1 M glycine (Sigma-Aldrich), pH 7.4. Cells were permeablized with PBS 0.2% (v/v) Triton X-100 (Sigma-Aldrich), stained for 30 min at 4 °C with Alexa fluor 555-phalloidin (Life Technologies) and washed with PBS containing 0.2% (v/v) Triton X-100 followed by PBS alone. In some experiments cell layers were also stained with polyclonal goat anti-VCAM-1 at 2 μg ml−1 (sc-1504, Santa Cruz) and detected with Donkey anti-goat Alexa Fluor 647 at 5 μg ml−1 (Jackson Immunoresearch, 705-606-147). Transwell membranes were transferred onto glass slides and visualized by confocal microscopy (Zeiss LSM 510 Meta, and LSM5 Duo) with a × 63 or × 40 objective, respectively. Z-stack images were acquired every 1 μm with 2-μm slice thicknesses for examination of in vitro endothelial monolayers. Images were analysed with Volocity version 6.1.1 software (Perkin Elmer).
Sc 1504
Manufactured by Santa Cruz Biotechnology
Sc-1504 is a piece of laboratory equipment produced by Santa Cruz Biotechnology. It is designed for use in scientific research and analysis. The core function of Sc-1504 is to provide a specific capability or measurement, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Donkey anti goat alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (1 mentions)
Lsm 5 duo, by Zeiss (1 mentions)
Volocity version 6, by PerkinElmer (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Ecl prime reagent, by GE Healthcare (1 mentions)
Adi spa 896, by Enzo Life Sciences (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Bx45 microscope, by Olympus (1 mentions)
Sc 1506, by Santa Cruz Biotechnology (1 mentions)
4 protocols using sc 1504
1
Visualizing T Cell Migration Across Endothelium
2
Quantification of HO-1 and VCAM-1 Proteins
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We separated HUVEC or cytotrophoblast protein lysates (20 μg) on 10% polyacrylamide gels with wet transfer to PVDF membranes (Millipore, Billerica, MA). We blocked membranes prior to incubation overnight with the primary antibody (Either anti-HO-1 at 1:500 dilution (ADI-SPA-896 (ENZO Life Sciences, Farmingdate, New York) or anti-VCAM 1 at 1:200 dilution (SC-1504), Santa Cruz, Biotechnology, Dallas, Texas)). We assessed heme‑oxygenase 1 (HO-1) by blocking blots with 5% skim milk followed by incubation with primary rabbit anti-HO-1 antibody (1:500) overnight at 4 °C, and incubated them with secondary anti-rabbit-HRP antibody for 1 h at room temperature. We examined vascular cell adhesion molecule 1 (VCAM 1) by blocking protein blots with 5% skim milk, followed by incubation with primary goat anti-VCAM 1 antibody (1:200) in Canget 1 overnight at 4 °C then incubated with secondary anti goat-HRP antibody for 1 h at room temperature. We applied ECL prime reagent (GE healthcare Life Sciences, Pittsburgh, PA) and immune-reactive bands were visualized using a Chemidoc XRS (BioRad, Hercules, CA). We stripped blots with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Waltham, MA) and re-probed it with mouse anti-B-actin (1:10,000) antibody overnight at 4 °C for the protein loading control.
3
Immunohistochemical Analysis of NIK and VCAM-1
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IHC was performed on the OCT sections using VECTASTAIN Elite ABC Kit (Vectorlabs). Briefly, air-dried slides were fixed in methanol containing 0.3% H2O2 for 10 min at -20°C, then serum-blocked for 20 min at room temperature, followed by antibodies incubation overnight at 4°C. Slides were washed three times with 1X PBS, then conjugated with secondary antibody and incubated for 1 hour at 37°C. After washing 3 times with PBS for 5 minutes, the tissues were stained with ABC reagent and DAB substrate according to manufacturer’s protocol. Slides were counterstained with hematoxylin. The antibodies were against NIK (Santa Cruz, sc-7211, 1:100), and VCAM-1 (Santa Cruz, sc1504, 1:100). We assessed the staining intensity with blinded genotype and treatment. Quantification was performed using the integrative optical density function in Image-Pro software.
4
Histological Analysis of Lung Inflammation
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Following BAL, the right lung lobes were fixed at 20 cmH 2 O with 10% buffered formalin, paraffin-embedded and cut into 4 µm sections. Sections were stained with hematoxylin and eosin [6] for scoring of pulmonary inflammatory cell infiltration and alveolar wall thickening using a semiquantitative score (0-3) on blinded, randomly selected images by two independent investigators [14] .
Similarly, formalin-fixed, paraffin-embedded sections were labelled with vascular cell adhesion molecule (VCAM)-1 antibody (C-19, Santa Cruz (sc-1504), 10 μg/ml) or PECAM-1 (M-20, Santa Cruz (sc-1506), 0.2 μg/ml) and counterstained with hematoxylin before visualisation by an Olympus BX45 microscope linked to a XC10 camera. Five images were collected per sample and were assessed using a semiquantitative score as above for intensity (0-3) and density of staining (0-3), on blinded sections by two independent investigators. To ensure specificity of the immunohistological staining reactions, consecutive sections were incubated in the absence of the primary antibody.
Similarly, formalin-fixed, paraffin-embedded sections were labelled with vascular cell adhesion molecule (VCAM)-1 antibody (C-19, Santa Cruz (sc-1504), 10 μg/ml) or PECAM-1 (M-20, Santa Cruz (sc-1506), 0.2 μg/ml) and counterstained with hematoxylin before visualisation by an Olympus BX45 microscope linked to a XC10 camera. Five images were collected per sample and were assessed using a semiquantitative score as above for intensity (0-3) and density of staining (0-3), on blinded sections by two independent investigators. To ensure specificity of the immunohistological staining reactions, consecutive sections were incubated in the absence of the primary antibody.
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