Midguts from Su(H)-lacZ; esg-Gal4,UAS-GFP/CyO female adults were dissected in cold phosphate-buffered saline (PBS) and fixed in PBS+4% formaldehyde and blocked in PBS Tween (PBS, 0.1% Triton X-100, and 1% bovine serum albumin). Antibodies included Chicken anti-GFP 1:4000 (Abcam, Cambridge, United Kingdom) and anti-phospho-Histone H3 1:100 (New England Biolabs, Ipswich, MA) and secondary antibodies AlexaFluor 488 goat anti-chicken and AlexaFluor 594 goat anti-rabbit IgG (Life Technologies, Eugene, OR) were used. Stained guts were mounted with Fluoroshield with DAPI (Sigma Aldrich) and Grace Bio-Labs SecureSeal imaging spacers. Samples were imaged using Perkin Elmer Spinning Disk confocal microscope with a 40× objective. Images were then processed using Image J.
Secureseal imaging spacer
Manufactured by Grace Bio-Labs
SecureSeal imaging spacers are designed to create a secure and consistent seal between microscope slide and coverslip during imaging procedures. The spacers maintain a fixed distance between the two surfaces, ensuring optimal sample imaging conditions.
Automatically generated - may contain errors
Lab products found in correlation
Spinning disk confocal microscope, by PerkinElmer (2 mentions)
Anti phospho histone h3, by New England Biolabs (2 mentions)
Chicken anti gfp, by Abcam (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Dichloromethane, by Avantor (1 mentions)
Potassium bicarbonate, by Merck Group (1 mentions)
Diethyl ether, by Thermo Fisher Scientific (1 mentions)
Irgacure 2959, by Merck Group (1 mentions)
Deuterated chloroform, by Avantor (1 mentions)
Acryloyl chloride, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Triethylamine, by Merck Group (1 mentions)
Sodium sulfate, by Merck Group (1 mentions)
Fluoroshieldtm with dapi, by Merck Group (1 mentions)
Molecular probes protein labeling kits, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 microscope, by Leica camera (1 mentions)
Ficoll 400, by Merck Group (1 mentions)
8 protocols using secureseal imaging spacer
1
Midgut Immunofluorescence Imaging Protocol
2
Isotopic Labeling of Drosophila Tissues
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Flies were transferred to a modified version of holidic medium (Kosakamoto et al., 2022 (link)) supplemented with 3.82 mM d8-Met (Cambridge Isotope Laboratories: DLM-6797–0.1) instead of Met, for up to 48 h prior to dissection unless otherwise stated. Third instar larvae or one-week-old adult Drosophila were placed in phosphate buffered saline (PBS) (4°C) and dissected using forceps (Dumont, Inox, #5) under a dissecting microscope. n = 3 or greater and carried out on different days using at least two different populations. Tissues were mounted on glass coverslips (Matsunami: (24 mm × 50 mm, 0.13–0.17 mm); (round, 12 mm, 0.13–0.17 mm)) with SecureSeal imaging spacers (Grace Bio-Labs, 654008), in PBS, PBS with 4% paraformaldehyde (adult gut only), or PBS with LysoTracker Deep Red (Invitrogen, L12492), 50 μM.
3
Synthesis of PEG-based Hydrogels
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Poly(ethylene glycol) (PEG, 2 kDa (Mn = 1917) and 10 kDa (Mn = 12 157)), acryloyl chloride, triethylamine, potassium bicarbonate, sodium sulfate and Irgacure 2959 were purchased from Sigma-Aldrich (St Louis, MO). Dichloromethane and deuterated chloroform with 0.03 vol% TMS were purchased from VWR Chemicals (Radnor, PA). Diethyl ether and Dulbecco's phosphate buffered saline were purchased from Fisher Scientific (Hampton, NH). SecureSeal Imaging Spacers were purchased from Grace Bio-Labs (Bend, OR). All reagents were used as received unless specified otherwise.
4
Dissection and Imaging of Drosophila Midguts
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Midguts from Su(H)-lacZ; esg-Gal4,UAS-GFP/CyO adults were dissected in cold PBS and fixed in PBS+4% formaldehyde and blocked in PBST (PBS, 0.1% Triton X-100 and 1% BSA). Antibodies included Chicken anti-GFP 1:4000 (Abcam, Cambridge, UK) and anti-Phospho-histone H3 1:100 (New England Biolabs, Ipswich, USA) and secondary antibodies AlexaFluor TM 488 goat anti-chicken and AlexaFluor TM 594 goat anti-rabbit IgG (Life Technologies, Oregon, USA) were used. Stained guts were mounted with Fluoroshield TM with DAPI (Sigma Aldrich) and Grace Bio-Labs SecureSeal™ imaging spacers. Samples were imaged using Perkin Elmer Spinning Disk confocal microscope with a 40X objective.
Images were then processed using Image J.
Images were then processed using Image J.
5
Visualizing Protein Phase Separation
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Purified proteins were diluted to desired protein and salt concentrations in 50 mM HEPES pH 7,5. Dilution of salt from 1 M NaCl (storage buffer) induced phase separation. A secure seal imaging spacer (Grace Biolabs) was used between slide and coverslip to visualize protein droplets in a Leica TCS SP8 microscope. Ficoll400 (Sigma) was used in addition to induce phase separation for DL2.
For DL2:hnRNPDL-Nt colocalization experiments, DL2 was green labeled with Oregon Green and hnRNPDL-Nt was red label with Texas Red using molecular probes protein labeling kits (Invitrogen) as described in the commercial protocol. A stock at 100 μM in 150 mM NaCl was prepared for each protein and then proteins were mixed at 1:1 ratio making posterior serial dilutions from the 50 μM stock mixture.
For RNA experiments, DL1 was used at 6.25 μM final concentration to obtain droplets of 2-6 μm and ensure that the surface was not fully covered with droplets to facilitate imaging. Total RNA was obtained from HeLa cells following the TRIzol Reagent user guide (Invitrogen). RNA diluted in RNase-free water was added at the indicated concentrations and posterior addition of 5 ng/μl of RNase (Thermo Scientific) was used to induce again LLPS.
For DL2:hnRNPDL-Nt colocalization experiments, DL2 was green labeled with Oregon Green and hnRNPDL-Nt was red label with Texas Red using molecular probes protein labeling kits (Invitrogen) as described in the commercial protocol. A stock at 100 μM in 150 mM NaCl was prepared for each protein and then proteins were mixed at 1:1 ratio making posterior serial dilutions from the 50 μM stock mixture.
For RNA experiments, DL1 was used at 6.25 μM final concentration to obtain droplets of 2-6 μm and ensure that the surface was not fully covered with droplets to facilitate imaging. Total RNA was obtained from HeLa cells following the TRIzol Reagent user guide (Invitrogen). RNA diluted in RNase-free water was added at the indicated concentrations and posterior addition of 5 ng/μl of RNase (Thermo Scientific) was used to induce again LLPS.
6
Thermal Dynamics of Pyruvate Decarboxylase
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Pyruvate decarboxylase (PDC) protein preps were diluted in buffer to 1 mg/mL concentration. 7.5 µL of protein solution was placed between two glass coverslips separated by a 0.15 mm deep SecureSeal imaging spacer (Grace Bio-labs, Bend, OR). Heating was controlled using a Linkam FTIR600 temperature-controlled microscope stage (Linkam Scientific Instruments, UK) and heated from 24 °C to either 60 or 120 °C at a ramp rate of 1 °C/min. The optics were set up for bright field differential interference contrast imaging on a NikonE800 microscope (Nikon, Tokyo, Japan), using a 20× 0.75 NA PlanApo ELWD objective. Images were captured every 30 s over the 3 h using a SPOT RTKE CCD camera (Diagnostic Instruments, Sterling Heights, MI) as TIFF stacks. TIFF stacks were analyzed using FIJI (ImageJ).
7
Sample Preparation for Microscopy
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A total of 8–9 µl of the fresh sample was loaded into a 120-µm-thick chamber built with two pieces of #1.5 cover glass with an imaging spacer (Grace Bio-Labs SecureSeal imaging spacer, 8 wells, diameter × thickness: 9 mm × 0.12 mm) in the middle.
8
Preparation of Dilute Gold Nanoparticle Suspension
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A volume of 1 mL stock gold colloids was centrifuged at 200× g for 30 min (Fisherbrand GT2R Centrifuge) for separation. After carefully removing the supernatant, the residue was re-dispersed in a surfactant solution using a vortex mixer. At this stage, the particle concentration was adjusted by diluting the particle–surfactant mixture with a particle-free surfactant solution of the same concentration to have a sparse appearance under an optical microscope and thus to ensure single-particle trapping. The approximate particle concentration was about particles/µL.
A fluid chamber was constructed using a pair of borosilicate glass coverslips (145 µm in thickness) and a double-sided adhesive spacer (120 µm in thickness, Grace Bio-Labs SecureSeal™ imaging spacer). We slightly overfilled the chamber with the dilute particle-surfactant mixture to minimize the air bubbles trapped inside the chamber. To prevent the liquids from evaporating, all trapping experiments were conducted within the fluid chamber. All glass coverslips were sonicated in acetone and isopropyl alcohol baths for 30 min each before use.
A fluid chamber was constructed using a pair of borosilicate glass coverslips (145 µm in thickness) and a double-sided adhesive spacer (120 µm in thickness, Grace Bio-Labs SecureSeal™ imaging spacer). We slightly overfilled the chamber with the dilute particle-surfactant mixture to minimize the air bubbles trapped inside the chamber. To prevent the liquids from evaporating, all trapping experiments were conducted within the fluid chamber. All glass coverslips were sonicated in acetone and isopropyl alcohol baths for 30 min each before use.
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