Alginate beads were fixed with 4% paraformaldehyde (PFA) in Tris-buffered saline (TBS) containing 5mM calcium chloride (CaCl2) at 4°C overnight and PFA was removed by washing with TBS containing 5 mM CaCl2. To stabilize bead structure during subsequent manipulations, beads were submerged in polyacrylamide solution (4% acrylamide and 0.13% bis-acrylamide, Bio-Rad Laboratories, Hercules, CA) at room temperature for 2 hours. The polyacrylamide solution was polymerized by adding 1/100 total volume of ammonium persulfate (10% w/v, APS) and 1/1000 total volume of tetramethylethylenediamine (TEMED) into the solution at room temperature for 1 hour. Beads were then submerged in 30% sucrose at 4 °C overnight, embedded in Tissue-Tek optimum cutting temperature embedding medium (Sakura Finetek USA Inc, Torrance, CA), and frozen at −80°C. Frozen blocks of beads were sectioned (60 μm thick) and stored on glass slides. Standard immunofluorescence protocol was conducted to stain Col VI of PCM using a Col VI primary antibody (Abcam, Cambridge, United Kingdom) and secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA).
Polyacrylamide solution
Manufactured by Bio-Rad
Sourced in United States
Polyacrylamide solution is a liquid polymer compound commonly used in electrophoresis applications. It serves as a matrix for the separation and analysis of biomolecules such as proteins and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Tissue tek optimum cutting temperature embedding medium, by Sakura Finetek (1 mentions)
Protein standard, by Bio-Rad (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Anti p38α, by Santa Cruz Biotechnology (1 mentions)
Anti ampkα, by Merck Group (1 mentions)
2 protocols using polyacrylamide solution
1
Encapsulation and Sectioning of Alginate Beads
2
Western Blot Analysis of Signaling Proteins
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Nitrocellulose membrane, polyacrylamide solution and protein standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Antibodies against phospho-extracellular signal-regulated protein kinase p-ERK [T202/Y204], phospho-AMP-activated protein kinase p-AMPK [T172], phospho-p38 mitogen-activated protein (MAP) kinase p-p38 [T180/Y182], phospho-ribosomal protein S6 p-S6 [S235/S236] and S6 were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-p70S6 kinase or 70 kDa ribosomal protein S6 kinase S6K1 [T389], anti-p38α and anti-ERKα antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-vinculin from Sigma Chemical Company (St Louis, MO, USA) and anti-AMPKα from Millipore (Paris, France). Alexa Fluor secondary antibodies were purchased from Molecular Probes (Invitrogen, Carlsbad, CA, USA).
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