Deconvolution imaging for synapse morphology was performed using a 100x (1.4 NA) plan Apochromat objective (Carl Zeiss) on an Axiovert 200 inverted microscope (Carl Zeiss) equipped with a cooled CCD camera (CoolSNAP HQ; Roper Scientific). Image acquisition and analysis were performed in SlideBook software (Intelligent Imaging Innovation). Maximum projections of deconvolved images were used for analyses. Quantification of Brp and bouton number was performed as previously described (Pielage et al., 2008 (link)). Confocal imaging was performed on a Yokagawa CSU22 spinning disk confocal with a 60x/1.4 plan Apochromat objective. Structured illumination fluorescence imaging for Endostatin-GFP and calcium channels was performed using an ELYRA PS.1 system (Carl Zeiss) with an inverted LSM 710 microscope equipped with a 63x(1.4 NA) plan Apochromat objective (Carl Zeiss) and an Andor iXon 885 EMCCD camera. For quantitative calcium channel abundance analysis, raw fluorescence intensities were preserved with position information using a customized algorithm (Carl Zeiss). See supplemental methods for details.
Plan apochromat objective
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat objective is a high-performance lens designed for microscopy applications. It features a planar image field and is corrected for chromatic and spherical aberrations, providing high-resolution, high-contrast imaging across a wide range of wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (24 mentions)
Axio observer z1, by Zeiss (18 mentions)
Lsm 880, by Zeiss (15 mentions)
Axiovert 200m, by Zeiss (10 mentions)
Lsm 780, by Zeiss (10 mentions)
Lsm 710 confocal microscope, by Zeiss (9 mentions)
Fm4 64, by Thermo Fisher Scientific (8 mentions)
Lsm 880 confocal microscope, by Zeiss (7 mentions)
Zen software, by Zeiss (6 mentions)
Lsm 710, by Zeiss (6 mentions)
Lsm 510 meta, by Zeiss (6 mentions)
Zen 2012, by Zeiss (5 mentions)
Lsm800 confocal microscope, by Zeiss (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Axio zoom v16, by Zeiss (4 mentions)
Axioplan 2 fluorescence microscope, by Zeiss (4 mentions)
Dp73 camera, by Olympus (3 mentions)
Zen blue 2011, by Zeiss (3 mentions)
Zen black software, by Zeiss (3 mentions)
Lsm710 pascal confocal microscope, by Zeiss (3 mentions)
157 protocols using plan apochromat objective
1
Synapse Morphology Deconvolution Imaging
2
Pyramidal Neuron 3D Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pyramidal neurons were selected for image acquisition based on their pyramidal or pear-shaped somata with 3–4 primary dendrites. Three-dimensional fluorescence images (16-bit, 512 × 512) were acquired using MetaMorph 7.0 (Universal Imaging) in conjunction with a Yokogawa CSU 10 spinning disk confocal (Perkin Elmer) fitted on a Zeiss Axiovert 200M inverted microscope. The excitation light of a Krypton/Argon ion laser (643-RYB-A02; Melles Griot) was selected by 488/10 nm, 568/10 nm or 647/10 nm filters (Sutter Lambda filter changer), reflected and then focused through a 63× 1.4 numerical aperture (NA) oil immersion Plan-Apochromat objective lens (Carl Zeiss MicroImaging, Inc.) or a 10× 0.45 NA Plan-Apochromat objective lens. Detection of the fluorescence emission, after passing a 525/50 nm bandpass filter for Alexa 488, a 607/45 nm bandpass filter for Alexa 568 or a 700/75 nm filter for Alexa 647, was obtained using a Cascade 512B camera (Roper). For between sample comparison, all images were acquired with the same settings without knowledge of the experimental condition during image acquisition. To acquire image stacks that could be deconvolved for further analysis, images were sampled using the Nyquist criterion.
3
Fluorescence Recovery After Photobleaching Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The inverse fluorescence after photobleaching setup has been described previously (van der Lelij et al, 2014 ). For photobleaching of mouse sororin‐LAP‐AID Cdca5 Δ/Δ, cells were imaged on a Zeiss LSM5 Duo confocal microscope using a 63× Plan‐Apochromat objective. Ten pre‐bleach images were acquired before bleaching a radial spot (r = 2 μm) three times at 100% laser intensity (100 mW diode 488) and acquiring 240 images at one‐second intervals. Photobleaching of N‐terminally tagged HeLa sororin, WAPL, and PDS5A cells was performed at the Stanford University School of Medicine Department of Biochemistry (USA) using a Nikon eclipse Ti microscope with an Apo TIRF 100× objective and equipped with an Andor iXon X3 camera and mosaic for spot bleaching, purchased using funds from a NIH S10 Shared Instrumentation Grant (S10RR026775‐01). Signal intensities were measured using ImageJ at bleached, nuclear and background regions, and normalized according to Ellenberg et al (1997 ). Data were analyzed using Berkeley Madonna software and curve fitting (a = (1−dS)*(1−EXP(−(kOff1)*time)) +dS*(1−EXP(−(kOff2)*time))).
4
Drosophila Synapse Immunostaining and Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Third instar larvae were dissected in HL3 buffer and subsequently fixed in HL3 + 3.7%PFA for 20 min. Tissue was permeabilized using 1× PBS with 0.2% Triton-X and 5% BSA. Staining was performed using the following probes/antibodies: horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, 1:250), Disc Large 1 (4F3, DHSB, 1:50), mouse monoclonal YARS1 (Abnova, 1:500), rabbit polyclonal GFP (Invitrogen, 1:2000), rabbit polyclonal RFP (Abcam, 1:250), mouse monoclonal Brp (nc82, DHSB, 1:100), mouse monoclonal FasII (1D4, DHSB, 1:250), mouse monoclonal Synapsin (SynORF1, 3C11, DHSB, 1:500). Alexa Fluor®−488 and Alexa Fluor®−546 secondary antibodies were used (Invitrogen, 1:1000). Muscle 6/7 of abdominal hemisegments 3 and 4 were imaged.
Laser scanning confocal microscopy was performed on a Carl Zeiss LSM700 microscope equipped with a 20× Plan-Apochromat (0.8 NA) or 63× Plan-Apochromat (1.4 NA) objective. Super-resolution structured illumination microscopy was performed on a Zeiss ELYRA S.1 microscope equipped with a 63× Plan-Apochromat objective (1.4 NA). For a description of methods used to calculate Lifeact-RFP distribution at synaptic boutons, please see Supplementary Fig.7 .
Laser scanning confocal microscopy was performed on a Carl Zeiss LSM700 microscope equipped with a 20× Plan-Apochromat (0.8 NA) or 63× Plan-Apochromat (1.4 NA) objective. Super-resolution structured illumination microscopy was performed on a Zeiss ELYRA S.1 microscope equipped with a 63× Plan-Apochromat objective (1.4 NA). For a description of methods used to calculate Lifeact-RFP distribution at synaptic boutons, please see Supplementary Fig.
5
Fluorescent and DIC Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dynamic fluorescent and differential interference contrast microscopy (DIC) was performed, and all images were collected using a Zeiss 510 Meta confocal microscope using a 63× Plan-Apochromat objective (Carl Zeiss, Jena, Germany). The images were extracted using the LSM browser (Carl Zeiss).
6
Immunostaining and Confocal Imaging of Mouse Brain
7
Immunostaining and Confocal Imaging of Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were anesthetized, rinsed by cardiac perfusion with 0.9% saline buffer and further fixed with 4% PFA. Whole brains were excised and post fixed in 4% PFA overnight, followed by cryoprotection treatment in 30% sucrose (wt/vol) in PBS for 16 h. Tissue blocks were then embedded in Optimal Cutting Temperature (OCT) compound and stored at −80°C. Briefly, 30 μm free-floating horizontal sections were washed three times with PBS and incubated at 4°C overnight on a rotator in 1 mL of primary antibody diluted in PBS containing 0.3% Triton X-100 (vol/vol) and 5% normal serum (vol/vol). After three washes with PBS-T (0.1% Triton X-100), the sections were incubated for 30 min with secondary antibodies (Molecular probes). Following three washes with PBS-T, and one wash with PBS, sections were mounted on slides. Fluorescent images were collected using a Zeiss LSM 700 META confocal microscope equipped with a 63× Plan-Apochromat objective and HeNE1, HeNe2 and argon lasers.
8
Automated Fluorescence Image Analysis
9
Cell Cycle Analysis via EdU Incorporation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To control for the non-dividing status of the confluent cell layer, cells were evaluated by eye and routinely tested by cell cycle analysis. For this, resting and exponentially growing cells were incubated for 3 h with 5 µM of the nucleotide analogue EdU. Cells were fixed using 3% para-formaldehyde and permeabilized with 0.2% Triton in PBS for 10 min at RT. EdU incorporated during the S phase of the cell cycle was stained via a click-it reaction according to the manufacturer’s instructions (PANATecs). DNA was stained with DAPI (0.2 µg/ml) for 5 min. Cells were washed again with ultrapure water, mounted with VECTASHIELD mounting medium (Vector Laboratories) and sealed with nail polish. The population was automatically scanned at a widefield microscope (Axiovert 200 M with a 20x Plan-Apochromat objective, Zeiss) and the EdU signal intensity was plotted vs. the DAPI signal intensity by using the program MetaCyte (MetaSystems). Non-dividing G0/G1 cells have a low EdU and a low DAPI signal (Fig. S1a,b ).
10
Quantifying Actin Filaments in Plant Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For in vivo microscopy, a Zeiss LSM 880 inverted confocal laser scanning microscope (Carl Zeiss AG, Germany) was used with either a 40× C-Apochromat objective (NA = 1.2 W) or a 20x Plan-Apochromat objective (NA = 0.8). GFP fluorescence (excitation 488 nm, emission 489–540 nm) was acquired in z-stacks (20–25 µm thickness). The maximum intensity projections obtained from the z-stacks were created using Zeiss ZEN Black software. Actin filaments density analysis was calculated by Fiji software (https://fiji.sc/ )42 (link) as the percent occupancy of GFP signal in each Maximum intensity projection. Image threshold was set to include all actin filaments and area fraction was measured. We analysed 7–11 cutouts from 6–7 plants for each variant. Representative images were selected from photos from at least 6 independent plants.
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!