To assess apoptosis in vivo 8 weeks after patch attachment, the animals were euthanized and TUNEL assays (Thermo Fisher Scientific) were performed. Briefly, five paraffin sections were deparaffinized and rehydrated, and the TUNEL assay was performed on 4-um-thick sections. Following permeabilization with 0.1% sodium citrate containing 0.5% Triton X-100 for 15 min, the reaction was carried out in a dark 37°C dry oven for 1 hour. Imaging of the heart sections was performed with an LSM 800 laser scanning microscope with Airyscan processing (Zeiss).
Lsm 800 laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 800 is a laser scanning microscope manufactured by Zeiss. It is designed to capture high-resolution, three-dimensional images of samples using a laser scanning technique. The microscope features a range of optical components and detectors to enable detailed analysis of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Airyscan processing, by Zeiss (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Hmvec c, by Lonza (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mouse anti tnnt2, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole solution, by Vector Laboratories (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
8 well glass chamber, by Thermo Fisher Scientific (2 mentions)
Accuri c6 plus flow cytometer, by BD (2 mentions)
Tunel assay, by Thermo Fisher Scientific (1 mentions)
Fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Faramount aqueous mounting medium, by Agilent Technologies (1 mentions)
Fcs express 6, by BD (1 mentions)
5 dodecanoylaminofluorescein di β d galactopyranoside c12fdg, by Thermo Fisher Scientific (1 mentions)
G3bp1, by Santa Cruz Biotechnology (1 mentions)
Pai 1, by Santa Cruz Biotechnology (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
35 protocols using lsm 800 laser scanning microscope
1
In Vivo Apoptosis Assessment
2
Uptake of Ac-SDKP by Human Cardiac Microvascular Endothelial Cells
3
Endogenous VAMP Localization in HUVEC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endogenous localization of the various VAMP proteins (VAMP 2/3/4/8) was determined using immunofluorescence. Shortly, HUVEC were grown on collagen‐coated 12 mm diameter coverslips. After reaching 70–80% confluency, cells were fixed in 4% PFA for 10 min at room temperature and permeabilized using 0.1% Triton‐X‐100 for 2 min. Cells were then blocked in 3% BSA in PBS for 30 min and subsequently stained with the respective VAMP or early endosomal antibodies for 1 h. Following PBS washes, the cells were stained with the corresponding fluorescent dye labeled secondary antibodies for 40 min and then mounted in mowiol. Imaging employed an LSM 800 laser scanning microscope (Zeiss) equipped with a 63x (NA 1.4) oil immersion objective (Plan‐Apochromat). Z‐stack projections of at least 10 cells were recorded per condition.
4
Immunofluorescence Analysis of DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated on poly-L-lysine coated coverslips and 48 h later, treated with AZD1775 and/or olaparib for 2 d, fixed in 3.7% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS (PBS-T). Cells were then treated with anti-RAD51 (cat.no.377467) and anti-γ-H2AX (cat.no.03-636), rinsed three times for 10 minutes in PBS, incubated with appropriate fluorophore-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA), and counterstained with DAPI (500 nmol/L, Invitrogen). Coverslips were then mounted onto slides using Faramount aqueous mounting medium (Dako, Glostrup, Denmark). Immunofluorescence was visualized using a Zeiss LSM 800 laser scanning microscope and photographs were taken at a magnification of X40.
5
Quantification of Hyaluronan Coverage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SN12L1 and HAS1−/− cells were immunostained for HABP as previously described [47] (link). Briefly, cells were fixed with 2% paraformaldehyde/0.1% glutaraldehyde for 30 min at room temperature and blocked with 2% goat serum for 30 min. Cells were then incubated overnight at 4 °C with biotinylated HABP. After washing 3× in PBS, cells were incubated with Alexa Fluor 488 anti-Biotin (Jackson ImmunoResearch Lab, West Grove, PA) and counterstained with DAPI. Cells were imaged with a Zeiss LSM 800 laser scanning microscope. HA coverage was determined using ImageJ software, as previously described [47] (link).
6
Cellular Uptake and Trafficking of Polymers
7
Quantifying Ac-SDKP Uptake in Cardiac Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine the uptake of Ac-SDKP by Human Cardiac Microvascular Endothelial Cells (HMVEC-Cs; Cat No. CC-7030, Lonza), we first performed in vitro cell imaging studies using fluorescein isothiocyanate (FITC) conjugated with Ac-SDKP. The FITC-conjugated Ac-SDKP was synthesized using Fmoc solid phase peptide synthesis (Genescript, Piscataway, NJ) and FITC-conjugated scrambled peptide, Ac-KDPS was synthesized using similar Fmoc synthesis protocol (Lifetein, Somerset, NJ). For these studies, peptide uptake was examined in both irradiated and non-irradiated (control) HMVEC-C cells at four hours post peptide treatment. For confocal image analysis, HMVEC-C cells were cultured on sterile cover slips in a 12 well plate. One of the plates was exposed to 9 Gy of radiation using a specialized orthovoltage radiation chamber and a sub group of cells were incubated with FITC-labeled Ac-SDKP or scrambled peptides at 14 μM. Images were captured using a confocal microscope (ZEISS LSM 800 Laser scanning microscope) at 400X magnification. For determining the uptake using FACs, cells were detached, irradiated and incubated with FITC-labeled scrambled peptide or Ac-SDKP (0.9, 1.8, 3.5, 7 & 14 μM). Cell uptake percentage was examined with a BD Accuri C6-Plus flow cytometer and further analyzed by FCS express 6-De Novo Software.
8
Quantifying Stress Granule Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IDH4 and WI‐38 cells grown on coverslips were fixed, and immunofluorescence experiments were performed as described 20 using antibodies against FMRP (1/6 dilution, kindly provided by Dr. R. Mazroui, Centre Hospitalier de l'Université Laval, Quebec, Canada), G3BP1 (1/1,000 dilution), PAI‐1 (1/250, Santa Cruz), and cyclin D1 (1/50, Abcam). In addition, immunofluorescence was performed to assess β‐galactosidase activity using 5‐dodecanoylaminofluorescein Di‐β‐D‐galactopyranoside (C12FDG) (ThermoFisher, D2893). Cells were treated for 1 h with bafilomycin A1 (Sigma‐Aldrich, B1793‐2UG), which disrupts lysosome formation leading to diffusion of β‐galactosidase into the cytosol, and then with C12FDG for 2 h. Before fixation, cells were treated with AS to induce SGs and immunofluorescence was performed using the Ziess Axio Observer.Z1 or the ZEISS LSM 800 Laser Scanning Microscope. The number of SGs was determined using ImageJ as previously described 77 .
9
Quantification of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RAD51 and γ-H2AX foci analysis, cells were seeded on a confocal dish, incubated for 48 hours, and then treated for 24 hours. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were incubated with a rabbit monoclonal RAD51 (#sc-398587) antibody and mouse monoclonal γ-H2AX (#05-636) antibodies overnight at 4°C. Samples were stained with fluorochrome-conjugated secondary antibody, Alexa Fluor 488 anti-rabbit (#2147635) and Alexa Fluor 594 anti-mouse (#2179228) (Invitrogen, Carlsbad, CA), and counterstained with DAPI (Sigma-Aldrich). Immunofluorescence was visualized using a Zeiss LSM 800 laser scanning microscope at 40× magnification.
10
Alkaline Comet Assay for DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Alkaline comet assays were conducted using a Trevigen Comet Assay kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Incorporated sybr-green was detected using a Zeiss LSM 800 laser scanning microscope. Photographs were taken at a magnification of X20, and tail intensities were measured using the Comet assay IV program (Andor Technology, Belfast, UK).
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!