EdU detection was performed using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen). Sections were permeabilized with 0.2% Triton X-100 (Progen Industries) in dPBS for 30 min, washed with 3% protease-free BSA (Sigma-Aldrich) in dPBS (2 × 5 min), then incubated with 300 μl reaction mixture prepared according to kit directions for 30 min. In the first pilot run, cells were not double labeled with Brn3a to distinguish RGCs, because at P0 there are as yet no displaced amacrine cells in the GCL (Perry, 1981 (link)). In the more extensive follow-up experiment we also wished to obtain laser-captured E18 RGCs from P5 retinas at a time when their axons are in the process of innervating the SC; however, at this age potentially EdU+ amacrine cells are now present in the GCL. Thus for all groups in this second series we additionally immunostained retinal sections for Brn3a protein, an established marker for RGCs projecting to the SC (Nadal-Nicolás et al., 2009, 2012 (link)). Retinal sections were washed with dPBS (2 × 10 min) and incubated overnight at 4°C with anti-Brn3a goat primary antibody (AB; Santa Cruz Biotechnology, SC-31984) 1:100 in antibody diluent (10% normal horse serum and 0.2% Triton X-100 in dPBS). After washes, sections were incubated in donkey anti-goat Cy3 (Jackson ImmunoResearch, 705-166-147; 1:200 in antibody diluent) for 2 h.
Protease free bsa
Manufactured by Merck Group
Sourced in Germany, United States
Protease-free Bovine Serum Albumin (BSA) is a highly purified protein preparation derived from bovine serum. It is characterized by the absence of detectable proteolytic activity, making it suitable for applications where the integrity of proteins or peptides is critical.
Automatically generated - may contain errors
Lab products found in correlation
Human plasmin, by Enzyme Research (3 mentions)
Tris hcl sds polyacrylamide gels, by Bio-Rad (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Triton x 100, by Santa Cruz Biotechnology (1 mentions)
Donkey anti goat cy3, by Jackson ImmunoResearch (1 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Cyclobutyl, by Cayman Chemical (1 mentions)
Cyclopentyl, by Cayman Chemical (1 mentions)
Cyclohexyl, by Cayman Chemical (1 mentions)
Digitonin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Dmem ham s f12, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Crif1, by Santa Cruz Biotechnology (1 mentions)
Anti cox 4, by Cell Signaling Technology (1 mentions)
Azure 300 chemiluminescent western blot imaging system, by Azure Biosystems (1 mentions)
6 protocols using protease free bsa
1
EdU Detection and Retinal Ganglion Cell Identification
2
Preparation of Cyclic Compound Standards
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Reference standards of cyclopropyl‐, cyclobutyl‐, cyclopentyl‐, cyclohexyl‐, and TMCPF were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The cell culture medium used was DMEM/Ham’s F12 with 15 mM HEPES, L‐glutamine, and without phenol red, from Thermo Fisher (Gothenburg, Sweden). Digitonin, ATP, trypsin, and protease‐free BSA were from Sigma‐Aldrich (Darmstadt, Germany). Fetal bovine serum (FBS) was from Life Technologies, Thermo Fisher (Gothenburg, Sweden). Coelenterazine was from Nanolight Tech (Pinetop, AZ, USA). Stock solutions of 500 μM coelenterazine were prepared in methanol (and protected from light), 50 mM Digitonin in DMSO, and 10 mM ATP in Milli‐Q water and stored at −20°C
3
Versatile Cell Infection Media Protocol
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Unless stated otherwise, the following media was used during virus infections on A549 cells: DMEM, 0.3% BSA (Equitech), 0.1% FBS, Pen/Strep, 1 μg/ml TPCK-trypsin. For infection of human fibroblasts, we used DMEM, 0.3% protease-free BSA (Sigma), 0.1% FBS, Pen/Strep, and 1μg/ml TPCK-trypsin. For infection in serum-free conditions on BHK cells, MEM with Pen/Strep was used, with or without addition of 1 μg/ml TPCK, as indicated. We used serum-free overlay for plaque assay on MDCK cells: DMEM, 0.00001% DEAE, 0.0001% NaHCO3, GlutaMax, NEAA, 0.5μg/ml TPCK, 1.2% avicel (or 1% oxoid agar).
4
Western Blot Analysis of Apoptotic Markers
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Tissues were collected and lysed in RIPA buffer (Sigma, St. Louis, MO, USA) in the presence of protease and phosphatase inhibitors (Halt™ protease and phosphatase inhibitor, Thermo Scientific, Waltham, MA, USA), and samples were prepared in protein sample buffer (0.25% bromophenol blue, 0.5M dithiothreitol, 50% glycerol, 10% sodium dodecyl sulfate (SDS), 0.25M Tris-Cl pH 6.8, and trace amounts of bromophenol blue), boiled, and stored at −80 °C. For gel electrophoresis and Western blot analyses, samples were run on 4–15% precast Tris-HCl SDS-polyacrylamide gels (BioRad, Hercules, CA, USA) and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Blots were successively probed with anti-COX4 (Cell signaling technology, #11967S), -CRIF1 (Santa Cruz, #sc-374122), -caspase 8 (Cell signaling technology, #4790S), -caspase 9 (Cell signaling technology, #9508S), -BCL-2 (Cell signaling technology, #2876S), or -β-actin (Cell signaling technology, #4967S) antibodies at 1:1000 dilutions in TBS containing 3% protease free BSA (Sigma, St. Louis, MO, USA). Blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA) and the images were acquired and quantitated using Azure 300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA).
5
Quantifying Growth Factor Release from Hydrogels
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To assess the ability to release growth factors, 50 uL HS (250 mM NaCl) and PS (145 mM NaCl) gels (10 mg/mL fibrinogen) were fabricated with 100 ng/mL FGF-2 (Peprotech, 100-18b) and 100 ng/mL VEGF-165 (Shenandoah Biotechnology, 100–44) in the bottom of 1.6 mL Eppendorf tubes. To fabricate these gels, a PBS solution of 400 ng/mL FGF-2 and 400 ng/mL VEGF-165 was used instead of the normal 4X cell solution in Step 3 of Section 2.1 above. After gelation, 1 mL of 0.5% protease-free BSA (Sigma, A3059) in PBS was added to each tube and was completely replaced daily. After 0, 1, and 7 days, the supernatant was removed and 50 uL of 0.2 mg/mL human plasmin (Enzyme Research Laboratories) in HEPES buffer (pH 8.5, Boston Bioproducts) was added. Fibrin degradation occurred overnight at 37°C. Growth factor retention was measured using ELISAs (Peprotech, 900-K08 and 900-K10) and a BioTek Synergy 2 luminescent plate reader (Gen5 software).
6
Protein Extraction and Western Blot Analysis
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Tissues were collected and lysed in RIPA buffer (Sigma) in the presence of protease and phosphatase inhibitors (Thermo Scientific), and samples were prepared in protein sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 1% β-mercaptoethanol and trace amounts of bromophenol blue), boiled, and stored at -80°C. For gel electrophoresis and western blot analyses, samples were run on 4–15% precast Tris-HCl SDS-polyacrylamide gels (BioRad) and transferred to polyvinylidenedifluoride (PVDF) membranes (BioRad). Blots were successively probed with anti-COX4 or -actin antibodies at 1∶1000 dilutions in TBS containing 3% protease free BSA (Sigma). Blots were visualized using SuperSignal chemiluminescence kits (Pierce Biotechnology) and the images were acquired and quantitated using FluorChem Q (Alpha Innotech).
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