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10 protocols using confocal lsm 510

1

Proximity Ligation Assay for ROCK1-APP and BACE1-APP Interactions

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Proximity ligation assay was performed using Duolink PLA Kit (Sigma‐Aldrich) in 4% PFA‐fixed brain tissue or cells before permeabilization in 0.1% Triton X‐100 for 30 min. The in situ PLA detection kit 596 was used to visualize single‐molecule interactions for ROCK1‐APP or BACE1‐APP. Samples were blocked and incubated with specific primary antibodies at 4°C overnight. Secondary antibodies (anti‐rabbit PLUS and anti‐mouse MINUS probes) were added to the reaction and incubated for 1 hr at 37°C. The hybridized oligonucleotides were ligated for 30 min at 37°C. Then, amplification solution was added with polymerase and incubated for 100 min at 37°C. After mounted with mounting medium, the PL signal was visualized as a chromatic (red) spot and was captured by Confocal LSM 510 (Zeiss).
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2

Immunofluorescent Staining of ID1 Protein

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Cells were stained with Phalloidin-fluorescein isothiocyanate (1:2, Sigma) and 4′,6-diamidino-2-phenylindole (1:20 000, ThermoFisher) and mounted with Mowiol (Vector). Primary antibody was anti-ID1 (1:500, rabbit monoclonal anti-mouse/human ID1, Biocheck, resp. rabbit polyclonal anti-mouse/human ID1, Abcam), the secondary species-matched antibody was conjugated to AlexaFluor 647 (1:500, anti-rabbit IgG Invitrogen). Images were acquired with confocal microscopy (Zeiss confocal LSM 510).
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3

Proximity Ligation Assay for STIM1-TRPC1 Interaction

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Freshly isolated VSMCs were studied using the Duolink in situ proximity ligation assay (PLA) detection kit 563 (Olink, Uppsala, Sweden) (Soderberg et al. 2008). Cells were adhered to coverslips, fixed in PBS containing 4% paraformaldehyde for 15 min, and permeabilized in PBS containing 0.1% Triton X‐100 for 15 min. Cells were blocked for 1 h at 37°C in blocking solution, and incubated overnight at 4°C with anti‐STIM1 and anti‐TRPC1 antibodies (both at dilution 1:200) in antibody diluent solution. Cells were labelled with combinations of either anti‐goat PLUS/anti‐rabbit MINUS or anti‐goat PLUS/anti‐mouse MINUS depending on animal species used for 1 h at 37°C. Hybridized oligonucleotides were ligated for 30 min at 37°C prior to amplification for 100 min at 37°C. Red fluorescence‐labelled oligonucleotides were then hybridized to rolling circle amplification products, and visualized using a Confocal LSM 510 (Carl Zeiss).
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4

Proximity Ligation Assay for TRPV4 Localization

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Freshly isolated ECs were studied using the Duolink in situ proximity ligation assay (PLA) detection kit 563 (Olink, Uppsala, Sweden) [50] (link). Cells were plated onto coverslips, fixed with PBS containing 4% paraformaldehyde for 15 min, and permeabilized in PBS containing 0.1% Triton X-100 for 15 min. Cells were blocked for 1 h at 37 °C in blocking solution, and incubated overnight at 4 °C with anti-TRPV4 and T1E3 antibodies (both at dilution 1:200) in antibody diluent solution. Cells were labelled with combinations of either anti-goat PLUS/anti-rabbit MINUS 1 h at 37 °C. Hybridized oligonucleotides were ligated for 30 min at 37 °C prior to amplification for 100 min at 37 °C. Red fluorescence-labelled oligonucleotides were then hybridized to rolling circle amplification products, and visualized using a Confocal LSM 510 (Carl Zeiss).
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5

Characterization of Blue-Emitting Quantum Dots

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UV–vis spectra were obtained
from the UV–vis spectrometer of Hewlett-Packard. PL spectra
were obtained from L550B luminescence spectrometer of Perkin-Elmer.
TEM and HRTEM images were recorded with FEI Tecnai T20 at an accelerating
voltage of 200 kV. AFM topographic images were obtained from XE-100
model of PSIA. FTIR spectra were obtained from Nicolet IR 200. XPS
data were obtained from Multilab 2000 of Thermo Scientific. Raman
spectra were obtained from LabRAM high-resolution dispersive Raman
microscope of Horiba Jobin Yvon using a 633 nm He–Ne laser
beam. The NMR spectra were recorded using an Avance digital 400 MHz
Bruker Spectrometer (Germany). ζ Potential measurements were
made with a Zetasizer ELS Z (Otsuka Electronics, South Korea). Confocal
fluorescent images were taken with a confocal laser microscopy system
(Confocal, LSM510, Carl Zeiss, Oberkochen 73447, Germany). The measurement
of QY of BPQDs and PEG-BPQDs was attempted by the reference point
method typically attempted for estimating QYs of other blue-emitting
fluorescent (nano)materials by a comparison with quinine sulfate (QY
of 0.54 in 0.1 M H2SO4).
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6

Visualization of Cilia and Sperm Structures

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Cells grown on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X‑100 in PBS, incubated in blocking buffer (2% heat‐inactivated goat serum, 0.2% Triton X‑100 in PBS) and probed with primary antibodies diluted in blocking buffer. After washing, appropriate secondary antibodies were added along with DAPI, before mounting in Mowiol. Cilia formation was induced by serum starvation for 24–48 h and visualized by anti‐acetylated tubulin, anti‐Arl13b or anti‐gamma‐tubulin antibodies. In some cases, cells were pre‐treated with different drugs for 10 h before staining. For analysis of the sperm, caudal epididymides from sexually mature males were finely minced and the sperm released into the buffer was collected and mounted. The slides were then air‐dried, fixed in 1.5% paraformaldehyde (v/v in PBS), permeabilized using 0.2% Triton X‐100, blocked in 2% BSA and stained with primary antibodies and DAPI. Fluorescence microscopy was performed using Zeiss Axioplan 2 Upright or Zeiss Confocal LSM 510 and analysed by using ImageJ software (https://imagej.nih.gov/ij/index.html).
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7

Impression Cytology for HLA-DR Expression

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IC was performed to evaluate the HLA-DR expression at the temporal bulbar conjunctiva, and was conducted 24 hours after the IVCM assessment to avoid misinterpretation due to the technical execution of the confocal microscopy. Samples were collected using Millicell-CM 0.4 µm (Millipore, Bedford, MA); the specimen acquiring procedure was performed as previously described.18 (link),19 (link) Zeiss Confocal LSM 510 (Carl Zeiss MicroImaging GmbH, Vertrieb, Germany) was used to visualize cells. Five different fields for each sample were evaluated. Positive (red nucleus and green cytoplasm) and negative (red nucleus) cells were counted. Minor irregularities that may have been present after cutting the membranes were normalized by reporting the percentage of positive cells for HLA-DR.
All evaluations of impression cytology samples were performed by two independent observers (authors L.B. and R.D.A.). IVCM (author M.L.) and IC (authors L.B. and R.D.A.) operators were masked for grouping and subject history of patients. Both eyes for patients were evaluated in the study, but one eye per subject was randomly chosen (using a computer-generated random number list) for the statistical analysis.
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8

Insulin Immunostaining in Islet Cells

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Confocal microscopy was performed as described previously (Aggarwal et al., 2011 (link)). NIT-1 cells and Mouse islets were plated on cover slips, fixed with 2 % formaldehyde, washed with PBS, permeabilized with 0.1% Trion–X100 and incubated with 2 % goat serum for one hour. The cells were then incubated with a primary antibody specific for Insulin (Cell Signaling Technology, Cambridge, MA) overnight at 4°C, then washed with PBS and incubated with a secondary antibody conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR) for one hour followed by three washings with PBS. Finally, cells were mounted with mounting medium from Molecular Probes (Carlsbad, CA) and images of insulin immunostained cells were acquired under an oil immersion objective (x40) with a confocal laser microscope (Zeiss Confocal LSM510, Carl Zeiss MicroImaging Inc.Thornwood, NY, USA) equipped with an argon-krypton laser.
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9

Immunostaining of C. elegans Embryos

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Immunostaining was performed using a methanol–4% formaldehyde fix as described previously (Skop and White, 1998 (link)) using the primary antibodies anti–ATX-2 mouse monoclonal (1:100; Ciosk et al., 2004 (link)), anti-GFP rabbit polyclonal (1:100; ab6556; Abcam, Cambridge, United Kingdom), anti–ZEN-4 rabbit serum (1:150; gift from Michael Glotzer, University of Chicago), and anti-actin [C4] mouse monoclonal (1:200; 691002; MP Biomedicals, Santa Ana, CA) diluted in PBSB (1× phosphate-buffered saline [PBS], 1% bovine serum albumin) and incubated overnight at 4°C. Unbound primary antibodies were removed by three washes for 5 min each with 1× PBS plus 0.5% Tween-20. Secondary antibodies used over the course of several different experiments were applied as follows: Alexa Fluor 488 anti-mouse (A-11001; Molecular Probes, Eugene, OR), Alexa Fluor 568 anti-mouse (A-11004; Molecular Probes), and Alexa Fluor 488 anti-rabbit (A-11008; Molecular Probes), with all secondary antibodies diluted 1:200 in PBSB. After 2 h of incubation in the dark at ambient conditions, unbound secondary antibodies were removed as described. The fixed and stained embryos were mounted with 8 μl of Vectashield with 4′,6-diamidino-2-phenylindole (DAPI; H-1200; Vector Laboratories, Burlingame, CA). Confocal imaging was performed on a departmental Zeiss 510 Confocal LSM operated with ZEN software (Carl Zeiss).
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10

Immunostaining of C. elegans Embryos

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Immunostaining was performed using a methanol-4%formaldehyde fix as previously described71 (link), and the following primary antibodies were used: anti-ATX-2 mouse monoclonal (1:100;Ciosk, 200421) and anti-GFP rabbit polyclonal (1:100; ab6556; Abcam) diluted in 1X PBS and 1% BSA (PBSB). Following an overnight incubation at 4°C, unbound primary antibodies were washed with PBST (1x PBS, 0.5% Tween). The secondary antibodies used were Alexa Fluor 568 anti-mouse (A-11004; Molecular Probes) and Alexa Fluor 488 anti-rabbit (A-11034; Molecular Probes) diluted 1:200 in PBSB. Secondary antibodies incubated at room temperature for 2 hours, and unbound secondary antibodies were removed with PBST washes. Embryos were mounted with Vectashield containing DAPI (H-1200; Vector Laboratories). Confocal imaging was performed on a departmental Zeiss 510 Confocal LSM operated with ZEN software (Carl Zeiss, Germany).
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