Immunofluorescence was performed, as previously described (51 (link)). Briefly, cells were grown overnight on glass coverslips, then fixed with 4% (wt/vol) paraformaldehyde (PFA) in PBS for 15 min at room temperature. Fixed cells were permeabilized/blocked with blocking solution [0.01% (wt/vol) Saponin (S7900; Sigma-Aldrich), 0.2% [wt/vol] pork skin gelatin (G2500; Sigma-Aldrich) in PBS] for 15 min at 37 °C. Next, cells were incubated with primary and, subsequently, with secondary antibodies for 1 h at 37 °C in blocking solution. Coverslips were mounted on slides with Fluoromount-G Mounting Medium (17,984–25; Electron Microscopy Sciences), and cells were imaged on a Zeiss confocal laser scanning microscope (LSM) 780 (Zeiss, Jena, Germany). Post-acquisition image processing was performed with ImageJ 1.36 (100 ). Co-localization analyses were performed with sets of images of the same cells (Z-stack) for each marker. Quantification was performed with ImageJ and the plug-in Co-localization Threshold to determine the Mander’s coefficients (tM) for each channel.
Pork skin gelatin
Manufactured by Merck Group
Sourced in United States
Pork skin gelatin is a gelling agent derived from the collagen found in pork skin. It is a water-soluble protein that can be used to thicken, stabilize, and add texture to a variety of food and pharmaceutical products.
Automatically generated - may contain errors
Lab products found in correlation
Saponin, by Merck Group (1 mentions)
Fluoromount g mounting medium, by Electron Microscopy Sciences (1 mentions)
Lsm 780 laser scanning confocal microscope, by Zeiss (1 mentions)
Basal medium 2 and supplement pack, by PromoCell (1 mentions)
Pump system, by Ibidi (1 mentions)
Glycerol, by Merck Group (1 mentions)
3 protocols using pork skin gelatin
1
Immunofluorescence Imaging and Colocalization Analysis
2
Laminar Flow Stimulates Endothelial Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3∗105 cells were seeded in EBM-2 full medium (Basal Medium 2 and Supplement Pack (PromoCell, no. C-22111) supplemented with 1% P/S) to ibidi μ-Slide I Luer 0.4 mm (ibidi, no. 80176) pre-coated with 0.1% pork skin gelatin (Sigma-Aldrich). For static conditions, cells were seeded to an equal area in a 10 cm dish, restricted by a silicone barrier during gelatin coating and cell seeding. Cells were kept under culture conditions (37°C, 5% CO2, 95% RH) for 48 h with daily medium exchanges. On the day of stimulation, medium was exchanged by pre-warmed EBM-2 starvation medium (Basal Medium 2 and Supplement Pack (PromoCell, no. C-22211) supplemented with 1% P/S and 2% FCS) and unidirectional laminar flow of 30 dyn/cm2 was applied for 2 h or 6 h (subsequent to a 5 h ramp phase to allow call adaption to increasing shear stress). Shear stress was applied using the ibidi pump system (ibidi GmbH) with the associated software (v 1.4.2).
For co-stimulation experiments, BMP9 in PBS was added to pump reservoirs and static controls dishes at a final concentration of 0.3 nM. Cells were then harvested for RNA and ATAC-seq (2 h) or protein analysis (6 h).
For co-stimulation experiments, BMP9 in PBS was added to pump reservoirs and static controls dishes at a final concentration of 0.3 nM. Cells were then harvested for RNA and ATAC-seq (2 h) or protein analysis (6 h).
3
Propolis-based Gelatin Film Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Propolis was obtained from an apiary located in a village in Mazowieckie Voivodeship (Wilkowyja, Poland) and frozen (-20 • C) until use. Pork skin gelatin (bloom = 175), glycerol (99% purity) and other used chemicals and reagents of analytical grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pork loins (M. longissimus thoracis et lumborum) were obtained from a local slaughter facility (Zakłady Mięsne Olewnik PLC, Sierpc, Poland) within two days of slaughter and transported in cold conditions to the Warsaw University of Life Sciences (Warsaw, Poland).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!