Bead aggregation assays were performed as previously described (Wang et al., 2003 (link), 2005 (link); Tang et al., 2016 (link)). In brief, purified GRASP65 full-length or N-terminal fragment was coupled onto the surface of Dynabeads M-500 Subcellular or M-450 Tosylactivated (Invitrogen). The coupled beads were then incubated with IC or purified proteins as indicated in the presence of an ARS at 37°C with rotation for 1 h. The beads were then submitted for imaging and quantitation. Random phase contrast digital images (10–20) of each sample were captured using Zeiss Observer Z1 epifluorescence microscope with a 10× lens. Images from three independent experiments were analyzed using MATLAB7.4 software to determine the surface area of objects, which was used to calculate the number of beads in the clusters. Aggregates were defined as those with six or more beads. For immunodepletion of DjA1, IC prepared from HeLa S3 cells (Rabouille et al., 1995 ) was incubated with nonspecific control IgG or DjA1 antibody (ProteinTech) and protein A-agarose beads at 4°C overnight. The beads were pelleted, and supernatant was analyzed by Western blotting of DjA1.
Observer z1 epifluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss Observer Z1 is an epifluorescence microscope designed for a wide range of applications. It provides high-quality imaging and illumination for fluorescence microscopy. The microscope features LED illumination, motorized components, and supports a variety of sample types and detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Prolong diamond antifade super resolution imaging mountant, by Thermo Fisher Scientific (2 mentions)
Tcs sp8 stimulated emission depletion sted super resolution microscope, by Leica (2 mentions)
Alexa fluor 488 labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
A1 confocal microsope, by Nikon (2 mentions)
Neutral red, by Merck Group (2 mentions)
Zen blue software, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Prolong antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Alexa 647 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Axiocam 506 mono, by Zeiss (1 mentions)
11 protocols using observer z1 epifluorescence microscope
1
Bead Aggregation Assay for GRASP65 Interactions
2
Immunofluorescence Microscopy for Golgi Structure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence microscopy was performed as previously described (Huang et al., 2016 (link)). Briefly, cells were rinsed with phosphate buffered saline (PBS), fixed with 4% (w/v) paraformaldehyde (PFA) for 15 min, and quenched with 50 mM NH4Cl for 10 min, followed by permeabilization with 0.2% (v/v) Triton X-100 in PBS for 10 min. Cells were then blocked with 1% (w/v) BSA Fraction V (Dot Scientific, DSA30075-100) for 1 h and incubated sequentially with a primary antibody and FITC- or TRITC-labeled secondary antibody diluted in 1% BSA in PBS (PBSB). DNA was stained with 1 μg/mL Hoechst 33342 (ThermoFisher). Coverslips were mounted on glass slides with Moviol and images were taken with a Zeiss Observer Z1 epifluorescence microscope with a 63× oil lens. For super-resolution microscopy, samples were prepared as previously described (Ireland et al., 2020 (link)). Briefly, Alexa Fluor 488-labeled secondary antibodies (ThermoFisher) were used. After washing, coverslips were mounted using ProLong Diamond antifade super-resolution imaging mountant (ThermoFisher). Super-resolution images were taken with a 100× oil lens on a Leica (Wetzlar, Germany) TCS SP8 STimulated Emission Depletion (STED) super-resolution microscope. Images were processed using the NIH ImageJ software. Brightness and contrast were adjusted linearly across all samples to clearly show the Golgi structure.
3
Visualizing SARS-CoV-2 RNA Interaction with TLR8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
moDCs were incubated with Atto-488–tagged SCV2-RNA1 (synthesized by Bio-Fab research) for 15 minutes, fixed with 4% paraformaldehyde (Pierce) for 10 minutes, and then seeded on glass slides by cytospin. After permeabilization with 100% cold methanol for 5 minutes, cells were labeled with a rabbit monoclonal anti–human TLR8 (11886, Cell Signaling Technology). A conjugate Alexa Fluor 594 anti-rabbit (A-11072, Thermo Fisher Scientific) was used as a secondary antibody. Glass slides were mounted using Prolong antifade with DAPI (Thermo Fisher Scientific). Cells were analyzed under a Zeiss Observer Z1 epifluorescence microscope equipped with a Plan-Apochromat 100×/1.4 numerical aperture oil objective and ApoTome2 imaging system for optical sectioning. Z-stack images were elaborated through AxioVision 3D and extended focus modules.
4
Mammary Gland Histological Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The carmine alumn stained whole mounts were prepared as described (52 (link)). The fourth inguinal mammary gland were fixed in 10% neutral buffered formalin for paraffin embedding and processed for immunohistochemistry as described (9 (link)). Oil Red O staining of cryosections of mammary glands was performed as described (53 (link)). Stained sections were imaged with a Observer Z1 epifluorescence microscope (Zeiss, Oberkochen, Germany) or with a A1 confocal microsope (Nikon Instruments Inc, Melville, NY).
5
Quantifying Immune Cell Activation by Imaging ASC Specks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following treatment, the cells were washed once in PBS, then fixed in 2% PFA at 4°C overnight (for BLaER1 ASC speck analysis) or 4% PFA on ice for 30 min. The cells were then washed twice in PBS containing 20 mM Glycine, then twice in PBS. To stain for intracellular targets, the cells were permeabilized in 0.1% Triton X-100 for 5 min and blocked in intracellular staining solution (PBS +10% goat serum, 1% HI-FBS, and 0.1% Triton X-100) for 30 min RT. Next, we used Alexa-647-conjugated Phalloidin (Invitrogen, A22287) for 30 min RT in an intracellular staining solution to stain actin. The cells were then washed (3× 5 min) and incubated with DAPI (1 μg/ml, 10 min) before being washed and imaged. For ASC speck detection, the fixed cells were incubated with DRAQ5 (eBioscience, 65-0880-96) for 5 min (1:2,000 dilution), then imaged directly. All imaging was performed with an Observer.Z1 epifluorescence microscope, 20× objective (dry, PlanApochromat, NA 0.8; ZEISS), Axiocam 506 mono, and ZEN Blue software (ZEISS). Image analysis of all ASC speck experiments was done using a cell profiler pipeline optimized to detect either ASC specks or nuclei. A minimum of 6 images was analyzed for each condition in each experiment.
6
Mammary Gland Histological Analysis
7
Immunofluorescence Microscopy for Golgi Structure
8
Mitotic Arrest and Golgi Fragmentation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitotic-arrested cells were first detached by washing off and washed five times with fresh growth medium without nocodazole; replaced into new dishes with polylysine-coated coverslips; and incubated for another 0.5, 1, 1.5, 2, and 3 h. Cells were then fixed in 3.7% paraformaldehyde, permeabilized with 0.3% Triton X-100, and processed for immunofluorescence microscopy with the indicated antibodies. Cells were observed using a 63× oil objective on a Zeiss Observer Z1 epifluorescence microscope. In interphase cells, fragmented Golgi was defined as disconnected or scattered dots. To quantify the percentage of cells with fragmented Golgi, we counted more than 300 cells in each treatment. The results are presented as the mean ± SEM from three independent experiments.
9
Immunofluorescence Staining of Cell Nuclei
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated on glass coverslips for 48 h, treated in 6-well plates, and then fixed in 3% formaldehyde for 20 min at room temperature. Cells were quenched with 50 mM NH4Cl in PBS, permeabilized for 5 min in 0.2% Triton X-100, and blocked with 3% BSA for 30 min. After washing, nuclei were stained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA, Cat #D1306) in PBS for 15 min before mounting according to the manufacturer’s instructions. Images were taken using a Zeiss Observer Z1 epifluorescence microscope with AxioVision Rel 4.8 software. DAPI produces a blue fluorescence when bound to DNA with excitation at 360 nm and emission at 460 nm. Specimens were stored at 4 °C. Experiments were prepared in triplicates.
10
Neutral Red Viability Assay for Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Neutral red stain is based on the ability of viable cells to incorporate and bind Neutral red dye in the lysosomes [91 (link)]. It was used to provide a qualitative estimation of the presence of viable cells in the fibroblast cell cultures.
Cells were cultivated in a 24-well cell culture plates and treated appropriately. The medium was removed from the fibroblast cell cultures and the cultures was washed with PBS. After that, 100 μL of Neutral red (Sigma-Aldrich, Saint Louis, MO, USA) dissolved in a cell culture medium (40 μg/mL) was added for each well, followed by a 4 h incubation at the appropriate culture conditions. After incubation, cells were gently washed twice with 150 μL of PBS. Images were taken using a Zeiss Observer Z1 epifluorescence microscope with AxioVision Rel 4.8 software.
Cells were cultivated in a 24-well cell culture plates and treated appropriately. The medium was removed from the fibroblast cell cultures and the cultures was washed with PBS. After that, 100 μL of Neutral red (Sigma-Aldrich, Saint Louis, MO, USA) dissolved in a cell culture medium (40 μg/mL) was added for each well, followed by a 4 h incubation at the appropriate culture conditions. After incubation, cells were gently washed twice with 150 μL of PBS. Images were taken using a Zeiss Observer Z1 epifluorescence microscope with AxioVision Rel 4.8 software.
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!