ABCB5+ DSCs, fibroblasts, keratinocytes and BM-MSCs were either lysed on ice for 10 minutes in RIPA Lysis Buffer for whole cell lysate (Santa Cruz Biotechnology) or cell lysis solution for extracellular matrix (ECM) protein extraction (5ml 1M NH4OH + 1.25ml Triton-100 + H2O to 250 mLs) at room temperature for one minute. ECM proteins were then dissolved in 4% SDS in 0.1M Tris·HCl. Equal quantities of protein (40 μg) were loaded onto a Novex WedgeWell 4–12% Tris-Glycine Mini Gels (Thermo Fisher). Following electrophoresis, protein was transferred onto a nitrocellulose membrane and incubated with anti-ABCB5 antibody (bs-1604R; Bioss), anti-C7antibody (sc-20774; Santa Cruz or gift from Dr. Woodley), anti-laminin alpha-3 (ab242197; Abcam), anti-laminin 332 (ab78286, Abcam), anti-laminin beta-3 (OTI3A2; Bio-rad) or anti-beta-actin antibody (A2547; Millipore Sigma) at 4°C overnight. Membranes were then incubated with goat anti-rabbit HRP-conjugated secondary antibody (sc-2004; Santa Cruz Biotechnology) or anti-mouse HRP-conjugated secondary antibody (sc-2005; Santa Cruz Biotechnology). Blots were developed using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher) for C7, laminin alpha-3, laminin 332 and laminin beta-3 or using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) for beta-actin and developed on X-ray film.
Ab242197
Manufactured by Abcam
Sourced in United States
Ab242197 is a lab equipment product. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab78286, by Abcam (1 mentions)
Anti mouse hrp conjugated secondary antibody sc 2005, by Santa Cruz Biotechnology (1 mentions)
Novex wedgewell 4 12 tris glycine mini gels, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp conjugated secondary antibody sc 2004, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
A2547, by Merck Group (1 mentions)
Ab97040, by Abcam (1 mentions)
Ix71 microscope system, by Olympus (1 mentions)
2 protocols using ab242197
1
Extracellular Matrix Protein Analysis
2
Immunohistochemical Analysis of LAMA3 Expression
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The expression of LAMA3 in 4-μm formalin-fixed paraffin-embedded (FFPE) sections was analyzed by IHC. IHC was conducted using the staining method based on standard peroxidase. The sections were incubated at 60°C for 1 h, deparaffinized in xylene for 3 × 10 min, and hydrated in gradient ethanol (100%, 100%, 95%, 90%, 80%, and 70%), followed by antigen retrieval by slide pretreatment in 0.01 M citrate buffer (pH = 6.0). Next, the sections were treated with 3% H2O2 for 10 min in order to block endogenous peroxidase. Subsequently, the sections were washed with 1 × PBS (pH = 7.4) and incubated with mouse antibody to LAMA3 (1:200, ab242197; Abcam, Cambridge, MA, USA) at 4°C overnight. Next, the sections were washed with 1 × PBS and incubated with goat anti-mouse immunoglobulin G (IgG; 1:500, ab97040; Abcam, Cambridge, MA, USA). The omission of primary antibody was taken as the NC for each sample. Finally, the immunoreactive products were visualized using 3,3-diaminobenzine. The results were evaluated using System Microscope IX71 (Olympus America, Center Valley, PA, USA).34 (link)
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