Immunohistochemistry was performed on 5μm paraffin sections as follows: paraffin sections were first warmed to 56°C in a vacuum incubator (Isotemp Vacuum Oven, Fisher Scientific) then washed immediately twice in xylene, gradually re-dehydrated in ethanol (100%, 95%, 70%, water), and then processed for antigen retrieval in citrate buffer (10mM pH6.0)/microwave (1000 watt, 6 minutes). Samples were then washed with PBS, blocked with 1% BSA/5% donkey-serum (1 hour, room temperature), then incubated overnight at 4°C with primary antibodies (1:200 dilutions in 0.5% BSA), washed 3 times with PBS, incubated with appropriate fluorescent labeled secondary antibodies (1:1000 dilution in 0.5% BSA, Life Technologies Inc) as well as the nuclear marker DAPI (Biolegend), and slides were then mounted using Gelvatol (Sigma-Aldrich) solution prior to imaging using a Zeiss LSM 710 Confocal microscope (Carl Zeiss, Jena, Germany) under appropriate filter sets.
Isotemp vacuum oven
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Isotemp Vacuum Oven is a laboratory equipment designed for drying, degassing, and heat treatment of samples. It provides a controlled environment with the capability to maintain precise temperatures and vacuum levels. The device offers reliable performance and consistent results for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Gelvatol, by Merck Group (2 mentions)
Lsm 710 confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (2 mentions)
Fluorescent labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Biocare Medical (1 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (1 mentions)
Glass vial, by Agilent Technologies (1 mentions)
Ammonium formate hcoonh4, by Merck Group (1 mentions)
Formic acid hcooh, by Merck Group (1 mentions)
24 r s hydroxycholesterol d6, by Avanti Polar Lipids (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Hexafluoro 2 propanol, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Wisent (1 mentions)
Fibrinogen, by Thermo Fisher Scientific (1 mentions)
Bovine fibrinogen, by Merck Group (1 mentions)
Tegaderm membrane, by 3M (1 mentions)
6 protocols using isotemp vacuum oven
1
Immunohistochemistry Protocol for Paraffin Sections
2
Immunofluorescent Staining of Lung Tissues
3
Quantification of 24S- and 27-Hydroxycholesterols
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All solvents and chemicals were HPLC grade. Methanol (CH3OH), ethanol (C2H5OH) and diethyl ether (CH3-CH2-O-CH2-CH3) were purchased from Fisher Scientific (USA), ammonium formate (HCOONH4) and formic acid (HCOOH) were purchased from Sigma Aldrich (St. Louis, MO). 24S-hydroxycholesterol and 27-hydroxycholesterol were purchased form Research Plus Inc. (Barnegat, NJ). Internal standard 24(R/S)-hydroxycholesterol (d6) [cholest-5-ene-3β, 24(R/S)-diol (d6)] was purchased from Avanti Polar Lipids (Alabaster, AL). Glass vials were purchased from Agilent Technologies, Inc. (Santa Clara, CA). Isotemp vacuum oven was purchased from Fisher Scientific (Model 285A, Pittsburgh, PA). Borosilicate-coated glass tubes and pipettes were used to reduce adhesion of sterols to plastic and glassware (Fisher Scientific, Pittsburgh, PA).
4
Preparation and Characterization of CNC Dispersions
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The
concentration of the as-received CNC dispersion was determined using
TGA in argon with a 10 °C/min ramp to 120 °C, followed by
an isothermal hold for 45 min. The initial dispersion was then diluted
to the desired concentration with ultrapure water (purified using
Labconco Water Pro BT, resistivity 18.2 MΩ cm at 25 °C,
pH 6.4). The mixture was vortexed for 2 min, followed by overnight
bottle rolling. The CNC-APTES dispersion was prepared similarly to
obtain the desired concentration. The dispersions were allowed to
rest for a minimum of 30 min to avoid bubbles in the cast films. The
shear cast films were prepared on a polyester substrate (0.005 in.
thick, Grainger) using a Gradco film applicator and MSK-AFA-II automatic
film coater (MTI Corporation). The speed was varied based on the wet
thickness to achieve a 15 s–1 shear rate. The wet
films were dried at 40 °C for 1 h (Isotemp Vacuum oven, Fisher
Scientific, Model 285A) and then overnight at room temperature (25
°C).
concentration of the as-received CNC dispersion was determined using
TGA in argon with a 10 °C/min ramp to 120 °C, followed by
an isothermal hold for 45 min. The initial dispersion was then diluted
to the desired concentration with ultrapure water (purified using
Labconco Water Pro BT, resistivity 18.2 MΩ cm at 25 °C,
pH 6.4). The mixture was vortexed for 2 min, followed by overnight
bottle rolling. The CNC-APTES dispersion was prepared similarly to
obtain the desired concentration. The dispersions were allowed to
rest for a minimum of 30 min to avoid bubbles in the cast films. The
shear cast films were prepared on a polyester substrate (0.005 in.
thick, Grainger) using a Gradco film applicator and MSK-AFA-II automatic
film coater (MTI Corporation). The speed was varied based on the wet
thickness to achieve a 15 s–1 shear rate. The wet
films were dried at 40 °C for 1 h (Isotemp Vacuum oven, Fisher
Scientific, Model 285A) and then overnight at room temperature (25
°C).
5
Fabrication of Multilayered Nanofibrous Scaffold
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Anionic dihydroxyl oligomer (ADO) and PU were synthesized as described previously.21 , 34 PU‐ADO (0.15% ADO by dry weight of PU) was dissolved in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (105 228, Sigma‐Aldrich, St. Louis, Missouri) to yield 18% concentration by dry weight of PU‐ADO. Aligned nanofibrous scaffolds were produced by electrospinning the polymer solution for 8 hours onto a 1150 rpm rotating mandrel (17 kV applied potential difference, 0.5 mL/hr infusion rate). Scaffolds were posttreated in an Isotemp Vacuum Oven (Model 281A, Fisher Scientific, Pittsburgh, Pennsylvania) overnight at 45°C. 3 × 50 mm scaffold strips were cut at a 30° angle relative to nanofiber direction, and sterilized by gamma irradiation (2.5 Mrad). Irradiated scaffold strips were soaked in 10 μg/mL fibronectin (f1141, Sigma‐Aldrich) in sterile phosphate buffered saline without Ca2+ or Mg2+ (PBS−/−; Wisent, St‐Bruno, Quebec) overnight at 37°C. Two fibronectin‐coated strips were stacked with opposing fiber angle orientation and rolled loosely around TYGON 3350 tubing (4 mm outer diameter; Saint‐Gobain, Beaverton, Michigan) to create individual six‐layered concentric constructs (Figure 2 ).
6
Preparation of Fibrin-Based Anisotropic Reinforcement Scaffolds
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