Laminin

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Laminin is a glycoprotein found in the extracellular matrix of cells. It is a major component of the basal lamina, which is part of the basement membrane. Laminin plays a crucial role in cellular attachment, migration, differentiation, and survival.

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15 protocols using laminin

Immunohistochemical Analysis of Neurovascular Markers

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For immunohistochemistry, cold saline of 10 ml and paraformaldehyde of 4% in 0.1 M PBS were used to anesthetize mice by perfusing through the heart. Then we harvested the brain quickly, and post-fixed in PFA of 4% for 48 h, and immersed in sucrose solution of 30%. They were storaged at 4 °C prior to be sectioned. Frozen sections were then cut by a thickness of 10 μm. Sections floating freely were washed by phosphate-buffered saline and blocked with Triton X-100 of 0.3% and normal blocking serum in PBS (10%) for 1 h at room temperature. Then they were stained overnight at 4 °C using the Claudin-5 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Occludin (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD13(1:50; Abcam, Cambridge, United Kingdom), Laminin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-amyloid (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody. The next day, PBS was used to wash sections and sections were incubated for 2 h with Cy3 conjugated anti-mouse IgG and Cy3 conjugated anti-rabbit IgG (1:100; Jackson immune Research Laboratories). Cells were stained by Occludin, Claudin-5, CD13, Laminin (red) or DAPI (blue). They were analyzed through an upright microscope (Ni-E, Nikon Corporation, Tokyo, Japan).

Lee M., Lee S.H., Kim M.S., Ahn K.S, & Kim M. (2021). Effect of Lactobacillus dominance modified by Korean Red Ginseng on the improvement of Alzheimer's disease in mice. Journal of Ginseng Research, 46(3), 464-472.

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Comprehensive Immunoblot Analysis of PTEN and Pathway

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PTEN (#9188S), Phospho-Serine (#9631S), GAPDH (#5174), Cleaved Caspase-3 (#9661), NOXA (#14766), BAX (#2772), PUMA (#4976) and HSP90 (#4874) were from Cell Signaling Technologies; PTEN (#sc133197), CK2 (#sc12738), Laminin (#sc-6216), USP7 (#sc30164), P21(#sc817), MDM2 (#sc965) were from Santa Cruz; Anti-Ubiquitin was from BD Pharmigen (#550944); GFP (A6455) was from Invitrogen; Tubulin (#T5201), Actin (#A5060) were from Sigma-Aldrich. HMG14 was from Abcam (#ab5212); pSer18 USP7 (#ABC225) was from Millipore. USP7 inhibitor (P5091), Idelalisib (CAL-101, GS-1101) and CK2 inhibitor (TBB) were from Selleckchem and Sigma-Aldrich, respectively.

Carrà G., Panuzzo C., Torti D., Parvis G., Crivellaro S., Familiari U., Volante M., Morena D., Lingua M.F., Brancaccio M., Guerrasio A., Pandolfi P.P., Saglio G., Taulli R, & Morotti A. (2017). Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/deleted clones. Oncotarget, 8(22), 35508-35522.

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Protein Extraction and Western Blot Analysis

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Cells were washed in ice-cold phosphate-buffered saline and lysed in T-PER (tissue protein extraction reagent) supplemented with Halt protease and phosphatase inhibitor cocktail (all from Pierce/Life Technologies, Grand Island, NY, USA). Protein content was measured by Bradford Assay; 30–50 μg protein was resolved on 12% SDS–PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in TBST (0.1% Tween-20 in TBS) and incubated overnight with the following antibodies: PAR1-ATAP2, EGFR, estrogen receptor, Laminin (Santa Cruz, Dallas, TX, USA), Vimentin V9, E-cadherin, Integrin α6, Keratin 8/18, Claudin 3 (Abcam, Cambridge, MA, USA), Zona Occluden-3 (Chemicon, Billerica, MA, USA), SLUG, Zeb1 (Cell Signaling Technology, Danvers, MA, USA).

Yang E., Cisowski J., Nguyen N., O’Callaghan K., Xu J., Agarwal A., Kuliopulos A, & Covic L. (2015). Dysregulated protease activated receptor 1 (PAR1) promotes metastatic phenotype in breast cancer through HMGA2. Oncogene, 35(12), 1529-1540.

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Fibroblast-Derived Neuronal Induction Protocol

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12‐well plates with or without coverslips were coated with 10 µg mL⁻¹ poly‐L‐lysine (PDL, Sigma, P6407) overnight, washed with sterile water 3 times, and then coated with 1 µg mL⁻¹ laminin (Santa Cruz, sc‐29012) overnight. Fibroblasts were seeded onto Fibronectin‐ or PDL/laminin‐coated plates and cultured in fibroblast medium for 24 h. The cells were transferred into neuronal induction medium with 20 µm (−)‐blebbistatin (Ble, MCE, HY‐13441) or the alternative active derivative, (S)‐(−)‐blebbistatin O‐benzoate (Ble‐OB, TRC, B208070) until cell confluence was about 40–60%. The medium containing Ble was changed every other day. After 7 days, the cells were switched to neuronal maturation medium or optimized maturation medium. Maturation medium was half‐changed every 3 days until the cells were subjected to further analysis.

He Z., Li Y., Feng G., Yuan X., Lu Z., Dai M., Hu Y., Zhang Y., Zhou Q, & Li W. (2022). Pharmacological Perturbation of Mechanical Contractility Enables Robust Transdifferentiation of Human Fibroblasts into Neurons. Advanced Science, 9(13), 2104682.

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Immunofluorescence Analysis of Muscle Proteins

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For immunofluorescence, 5 μm thick paraffin sections of vastus lateralis were re-hydrated and antigens were retrieved. Sections were then permeabilized with Triton X-100 0.5% in PBS for 10 minutes at RT and non-specific interactions were blocked with 10% HS in PBS for 30 minutes at RT. After washing in PBS tissues were incubated with primary antibody against FNDC5 (Abcam), ATPsynthase (Invitrogen Molecular Probes), Laminin (Santa Cruz), MyoD (Dako) and Pax7 (R&D Systems). Fluorescent-labeled secondary antibodies were anti-mouse Alexa Fluor®-555 and Alexa Fluor®-594; anti-rabbit Alexa Fluor®-488 and Alexa Fluor®-594; anti-rat Alexa Fluor®-568 (Thermo Fisher Scientific). Nuclei were counterstained with fluorescent mounting medium plus 100 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). At least 5 high power fields were analyzed for each section. Staining was never observed when the primary antibody was omitted. Percentage of positive fibers were calculated by using the NIS-Element BR 4.10.00 software.

Colaianni G., Mongelli T., Cuscito C., Pignataro P., Lippo L., Spiro G., Notarnicola A., Severi I., Passeri G., Mori G., Brunetti G., Moretti B., Tarantino U., Colucci S.C., Reseland J.E., Vettor R., Cinti S, & Grano M. (2017). Irisin prevents and restores bone loss and muscle atrophy in hind-limb suspended mice. Scientific Reports, 7, 2811.

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Spinal Cord Histological Analysis

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Spinal cords were dissected, fixed overnight in 4% paraformaldehyde at 4°C, and then transferred to 30% sucrose for 72 hours. Longitudinal sections of spinal cords containing the lesion/graft site were sectioned. Blocking was done with 5% donkey serum, spinal cord sections were stained for vascular endothelial-Cadherin (1:200, Santa Curz), Laminin (1:200, Santa Cruz, Dallas, TX), Beta-III tubulin (1:200, R&D Systems, Minneapolis, MN), Nestin (1:200, Abcam, Cambridge, MA), glial fibrillary acidic protein (GFAP) (1:200 Abcam), CSPG (1:200, Sigma), and with secondary antibodies (1:200, Invitrogen, Carlsbad, CA).

Marrotte E.J., Johnson K., Schweller R.M., Chapla R., Mace B.E., Laskowitz D.T, & West J.L. (2021). Induction of Neurogenesis and Angiogenesis in a Rat Hemisection Spinal Cord Injury Model With Combined Neural Stem Cell, Endothelial Progenitor Cell, and Biomimetic Hydrogel Matrix Therapy. Critical Care Explorations, 3(6), e0436.

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Immunohistochemical Analysis of Placental Laminin

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Paraffin sections of the placenta were deparaffinized, rehydrated, and subjected to antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven. Then, the sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. The placental sections were subsequently incubated with primary polyclonal antibodies against laminin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted 1 : 100 and 1 : 50. After washing with tris-buffered saline (TBS), specific secondary antibodies were applied, and then, the sections were incubated with an avidin-biotin-peroxidase complex according to the manufacturer's instructions (ABC-peroxidase kit, Vector Labs, Burlingame, CA, USA). Finally, the sections were incubated in a solution of 0.05% 3–3′-diaminobenzidine tetrahydrochloride (Sigma Aldrich, St. Louis, MO, USA) and 0.33% hydrogen peroxide. All sections were counterstained with Harris haematoxylin, dehydrated in a graded alcohol series and in xylene, and coverslipped.

Wang F., Cao G., Yi W., Li L, & Cao X. (2019). Effect of Metformin on a Preeclampsia-Like Mouse Model Induced by High-Fat Diet. BioMed Research International, 2019, 6547019.

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Cardiac Fibroblast-Myocyte Co-culture

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Glass bottom dishes of 3.5 cm were coated with laminin (Santa Cruz, sc-29012) at room temperature for 45 min. 2.5E5 fibroblasts senesced via etoposide treatment were seeded on 3.5 cm laminin-coated glass bottom dishes a day before the addition of cardiomyocytes. 1.25E5 proliferating fibroblasts were seeded on glass bottom dishes a day before cardiomyocyte seeding to allow to double once in order to reach approximately 2.5E5 cells. DMEM/F-12 (Life Technologies) supplemented with penicillin (100 µg/mL) and streptomycin (100 µg/mL) was used for fibroblasts until the addition of myocytes. Freshly isolated 4-month-old rabbit cardiomyocytes were allowed to gravity pellet for 30 min. The supernatant was removed, and cells were resuspended in serum-free myocyte media (Medium 199, Earle’s Salts, Thermo Fisher Scientific). Finally, fibroblast media was removed, and cardiomyocytes were added to dishes containing fibroblasts with 6.2E4 live myocytes per dish. Dishes were kept at 37°C for 24 hr before patch clamping.

Baggett B.C., Murphy K.R., Sengun E., Mi E., Cao Y., Turan N.N., Lu Y., Schofield L., Kim T.Y., Kabakov A.Y., Bronk P., Qu Z., Camelliti P., Dubielecka P., Terentyev D., del Monte F., Choi B.R., Sedivy J, & Koren G. (2023). Myofibroblast senescence promotes arrhythmogenic remodeling in the aged infarcted rabbit heart. eLife, 12, e84088.

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Immunofluorescence Labeling of Stem Cells

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hPDLSCs placed on CTRL and TEST samples were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde in 0.1 M PBS, pH 7.4. After PBS wash, cultures were made for immunofluorescence labelling. Then, cells seeded on granules were permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 5% skimmed milk in PBS. Primary monoclonal antibodies to anti-human Fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Laminin (Santa Cruz Biotechnology), N-Cadherin (Santa Cruz Biotechnology) and RUNX2 (Santa Cruz Biotechnology) were utilized, followed by Alexa Fluor 488 green fluorescence conjugated goat anti-mouse as secondary antibodies (Molecular Probes, Invitrogen, Eugene, OR, USA). Then, the specimens were incubated with Alexa Fluor 594 phalloidin red fluorescence conjugate (Molecular Probes) to stain actin cytoskeleton. Nuclei were dyed with TOPRO (Molecular Probes). Specimens were positioned facing down on glass slides and mounted with Prolong antifade (Molecular Probes) [27 (link)]. The stained samples were evaluated using a Zeiss LSM800 META confocal system, connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective (40×/1.3 NA). The images were taken using an argon laser beam with excitation lines at 488 nm.

Marconi G.D., Fonticoli L., Della Rocca Y., Rajan T.S., Piattelli A., Trubiani O., Pizzicannella J, & Diomede F. (2021). Human Periodontal Ligament Stem Cells Response to Titanium Implant Surface: Extracellular Matrix Deposition. Biology, 10(9), 931.

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Wound Healing Assay of iPS-RPE Cells

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The iPS-RPE cells were cultured on 24-well plates coated with either matrigel, fibronectin (5 µg/cm²; Santa Cruz Biotechnology, Dallas, TX), laminin (5 µg/cm²; Santa Cruz Biotechnologies), or collagen IV (5 µg/cm²; Santa Cruz Biotechnologies) for 8 weeks. A wound approximately 500 µm wide was created by scratching the cell layer with a sterile 1000 µL pipette tip. Cells were then washed twice with DPBS (Dulbecco's phosphate-buffered saline) to remove detached cells and cultured for up to 1 month, with images of the wound recorded regularly using a Nikon Diaphot inverted light microscope (Nikon, Tokyo, Japan). The average size of the wound was analyzed by recording five separate fields per well and calculating the wound area in ImageJ,²⁸ (link) either using the MRI Wound Healing Tool (available at http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool) or by manually tracing the wound.

Carlsson E., Supharattanasitthi W., Jackson M, & Paraoan L. (2020). Increased Rate of Retinal Pigment Epithelial Cell Migration and Pro-Angiogenic Potential Ensuing From Reduced Cystatin C Expression. Investigative Ophthalmology & Visual Science, 61(2), 9.

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