Morphological observation of root tip cells was carried out with 100 μg/mL propidium iodide (PI) solution and photographed using a laser confocal microscope (Leica LAS SP8).
Las sp8
Manufactured by Leica
The LAS SP8 is a confocal laser scanning microscope produced by Leica. It is designed for high-resolution imaging of biological and material samples. The core function of the LAS SP8 is to provide advanced optical sectioning and 3D imaging capabilities through the use of a scanning laser and sensitive detectors.
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Lab products found in correlation
Application suite advanced fluorescence las af software, by Leica (3 mentions)
Z 1 light sheet microscope, by Zeiss (1 mentions)
Fv10 asw 4, by Olympus (1 mentions)
Rabbit antibody to ki67, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mouse antibody to tuj1, by Fortrea (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Rabbit anti h3k27me3 antibody, by Merck Group (1 mentions)
7 protocols using las sp8
1
Morphological Observation of Root Tip Cells
2
Acceptor Photobleaching FRET Imaging
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For BiFC-FRET assay, 16 h (HEK 293 cells) or 24 h (neuro-2a cells) after transfection, cells were fixed with PBS/2% paraformaldehyde, washed and mounted on slides for FRET detection. Samples were then subjected to acceptor photobleaching FRET imaging under a confocal microscope (LAS SP8; Leica) with a 63×/1.40 NA oil objective (Leica) as described previously (Wang et al., 2015 (link)). Image acquisition, registration, background subtraction, and data analyses were performed with Leica Application Suite Advanced Fluorescence (LAS AF) software. Imaging conditions were set up manually: mTurquoise2 (excitation: 458 nm, emission: 465–505 nm), and Venus (excitation: 514 nm, emission: 525–600 nm). Photobleach was performed using acceptor excitation light and the similar bleach efficiency (~80%–90%) was achieved. Images of donor and acceptor channels were acquired pre- and post-bleach, respectively. FRET efficiency was calculated as percentage of enhancement in donor fluorescence (f) after acceptor photobleaching: E = 1 − f[CFP(pre)]/f[CFP(post)]. Five non-bleached regions were selected and the average value was used to correct the FRET efficiency of photobleached region.
3
Immunostaining of Human Neural Stem Cells
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For immunostaining, human NSCs or hiPSCs were seeded in 24‐well culture dishes with a 1 × 1 cm diameter glass coverslip in each well. After infection or treatment, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 10 minutes and permeabilized with 0.3% Triton X‐100 in PBS for 15 minutes before being treated with 5% bovine serum albumin (BSA) for 1 hour at RT. Cells were incubated overnight at 4°C with various combinations of the following primary antibodies: rabbit antibody to Ki67 (Abcam, 1:500), rabbit antibody to Nestin (Millipore, 1:500), mouse antibody to Tuj1 (Covance, 1:500), goat antibody to Sox2 (R&D, 1:300), rabbit antibody to Oct4(Abcam, 1:500), or goat antibody to Sox1(R&D, 1:300). After washing with PBS/1% BSA three times, cells were incubated with Alexa Fluor 594‐labelled donkey anti‐goat IgG, Alexa Fluor 647‐labelled donkey anti‐rabbit IgG or Alexa Fluor 488‐labelled donkey anti‐mouse secondary antibodies in the dark for 1 hour. Followed by washing with PBS/1% BSA, cells were stained with DAPI (Sigma) and mounted on slides. Images were acquired under microscope (LAS SP8; Leica, FV10‐ASW 4.2; Olympus, Zeiss Z1 and Zeiss Z.1 Lightsheet microscope; Zeiss). A Canny Edge Detection plugin of ImageJ was used to measure fold density in images of Hoechst‐stained organoids by stereoscopic microscope.
4
Acceptor Photobleaching FRET Imaging of HEK293 Cells
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HEK293 cells were cultured on glass coverslips and transfected with Effectene Transfection Reagent (QIAGEN, Hilden, Germany). After fixing in 2% paraformaldehyde in PBS for 30 min, cells were washed in PBS three times and mounted on slides. Samples were subjected to acceptor photobleaching FRET imaging with a confocal microscope (LAS SP8; Leica) with a 63×/1.40 NA oil objective (Leica). Image acquisition, registration, background subtraction and data analyses were performed with Leica Application Suite Advanced Fluorescence (LAS AF) software. Imaging conditions were set up manually: CFP (excitation: 405 nm, emission: 465–505 nm) and YFP (excitation: 514 nm, emission: 525–600 nm). Photobleach was performed using 514-nm light and over 70% bleach efficiency was achieved. Images of CFP and YFP channels were acquired pre- and post-bleach. FRET efficiency was calculated as percentage of enhancement in donor fluorescence (f) after acceptor photobleaching: E=1-f(CFP(pre))/f(CEP(post)). Five non-bleached regions were selected and the average values were used to correct the FRET efficiency of photobleached region.
5
Quantifying Bimolecular Fluorescence Complementation in HEK 293 Cells
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HEK 293 cells were transfected with BiFC constructs for 16 h, fixed with PBS/2% paraformaldehyde, stained with Hoechst 33342 and then visualized by confocal microscopy (LAS SP8; Leica) using 20×/0.75 NA IMM or 63×/1.40 NA oil objective (Leica). Imaging conditions were used: Hoechst 33342 (excitation: 405 nm, emission: 425–465 nm), and Venus (excitation: 514 nm, emission: 525–600 nm). Images acquired under 20× objective were used for quantification.
To monitor the expression of BiFC constructs in cells, HEK 293 cells used for Western blots analysis were plated and transfected at the same time with the same transfection mix as BiFC analysis. HEK 293 cells were also harvested 16 h after transfection for immunoblots analysis.
The fluorescence intensity of Venus and total cell number in 20× images and protein expression analyzed by Western blots were quantified respectively using ImageJ software, and relative BiFC signal was calculated as: Venus fluorescence intensity/(total cell number × expression of ADAM10 and BACE1).
To monitor the expression of BiFC constructs in cells, HEK 293 cells used for Western blots analysis were plated and transfected at the same time with the same transfection mix as BiFC analysis. HEK 293 cells were also harvested 16 h after transfection for immunoblots analysis.
The fluorescence intensity of Venus and total cell number in 20× images and protein expression analyzed by Western blots were quantified respectively using ImageJ software, and relative BiFC signal was calculated as: Venus fluorescence intensity/(total cell number × expression of ADAM10 and BACE1).
6
Immunostaining of Drosophila Wing Discs
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3rd instar larvae wing discs were dissected for immunostaining using the standard protocol as reported previously73 (link),74 (link). Larvae with indicated genotypes were fixed in 4% formaldehyde in PBS buffer, washed and incubated with a specific combination of primary and secondary antibodies in PBS/0.1% Triton X-100, and mounted in 40% glycerol. Pictures were taken with the confocal microscope (LAS SP8; Leica) using a 40×/1.25 NA oil objective (Leica). Antibodies used in this study were rat anti-Ci antibody (DSHB), rabbit anti-Pc antibody (which was generated in this study by using the peptide RKAEVLKESGKIG), mouse anti-Ubx antibody (DSHB), guinea pig anti-Sens antibody (a kind gift from Dr Hugo J Bellen), rabbit anti-H3K27me3 antibody (Millipore), mouse anti-CD2 antibody (AbD Serotec), rabbit anti-H4K20me1 antibody (Abcam), and secondary antibodies were bought from Millipore.
7
Acceptor Photobleaching FRET Assay
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The assay was performed following the reported methods [28 (link), 29 ]. HEK293 cells were seeded in 24-well plate with cover-slips and transfected. After 48 h, cells were treated as indicated and fixed with 4% PFA for 10 min, washed with PBS for three times, and mounted on slides. Samples were subjected to acceptor photobleaching FRET imaging with a confocal microscope (LAS SP8; Leica) with a 63×/1.40 NA oil objective (Leica). Image acquisition, registration, background subtraction and data analyses were performed with Leica Application Suite Advanced Fluorescence (LAS AF) software. Imaging conditions were set up manually: CFP (excitation: 405 nm, emission: 465–505 nm) and YFP (excitation: 514 nm, emission: 525–600 nm). Photobleach was performed using 514-nm light and over 70% bleach efficiency was achieved. Images of CFP and YFP channels were acquired pre- and post-bleaching. FRET efficiency was calculated as percentage of enhancement in donor fluorescence (f) after acceptor photobleaching:
E = 1-f[CFP(pre)] / f[CEP(post)]. Five non-bleached regions were selected and the average values were used to correct the FRET efficiency of photobleached region.
E = 1-f[CFP(pre)] / f[CEP(post)]. Five non-bleached regions were selected and the average values were used to correct the FRET efficiency of photobleached region.
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