Primary human lung fibroblasts overexpressing flotillin-2–Myc or LacZ were fixed with cold 2% paraformaldehyde in PBS for 15 min. For localization of Endo180 and FLOT2, the internalized collagen study, primary human lung fibroblasts were incubated with FITC-conjugated collagen type I (25 μg/ml final concentration; Invitrogen) for 3 h at 37°C before fixation. Cells then were permeabilized with 0.1% Triton-X 100 in PBS (PBS-T) for 10 min, blocked with 5% normal goat serum in PBS-T, and incubated with primary antibodies to Myc (1:100) and Endo180 (1:50) or FLOT2 (1:100) and Endo180 (1:50) overnight at 4°C, followed by Alexa Fluor 488– and Alexa Fluor 594– or Alexa Fluor 647– and Alexa Fluor 594–conjugated secondary antibodies (1:100; Invitrogen) for 1 h at room temperature. Confocal images were captured by a Leica point scanner using a Leica TCS SPE confocal microscope with an ACS APO 63× Oil CS objective lens at room temperature with Leica Type F Immersion Liquid (11513859). Images were processed with Photoshop CS6 software (Adobe, San Jose, CA).
Type f immersion liquid
Manufactured by Leica
Sourced in Japan, Germany
The Type F immersion liquid is a specialized product designed for use with Leica microscopes. It serves as a high-refractive-index immersion medium, optimized for enhancing image quality and resolution during microscopic observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 confocal microscope, by Leica (2 mentions)
Ultracomp beads, by Thermo Fisher Scientific (2 mentions)
Channel dye separation module, by Leica (2 mentions)
Dm6000, by Leica (2 mentions)
Tcs sp8, by Leica (2 mentions)
Fitc conjugated collagen type 1, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 sted 3x microscope, by Leica (1 mentions)
Mouse anti flag m2 antibody, by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Las x software, by Leica (1 mentions)
Dmem without phenol red, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 system, by Leica (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Alexa fluor 555 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Eclipse te2000 u fluorescent microscope, by Nikon (1 mentions)
El6000, by Leica (1 mentions)
Dfc350 fx camera, by Leica (1 mentions)
Vectashield4 6 diamidino 2 phenylindole dapi mounting medium, by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
11 protocols using type f immersion liquid
1
Collagen Internalization in Lung Fibroblasts
2
STED Microscopy of Fluorescent Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
STED images were acquired with a Leica TCS SP8 STED 3X microscope (equipped with white-light laser, 405-nm diode laser and HyD detectors) (Leica Biosystems, Concord, ON, Canada) by sequential scanning between stacks with a 592-nm (Alexa Fluor 488) or 660-nm (mCherry) depletion using the following settings: objective, HC PL/APO CS2 ×100/1.40 oil STED White; immersion oil, Leica Type F Immersion liquid (refractive index, 1.5180); zoom factor, 6.00; scan speed, 600 Hz; line average, 8 to 16 (Alexa Fluor 488 or mCherry, respectively); time gate, 0 to 3.5 ns; STED 3D, 50%; depletion laser intensity, 5% to 10% (Alexa Fluor 488, depending on the signal) or 30% (mCherry). Laser power and gain were set to optimize signal-to-noise ratio and avoid saturation using the QLUT Glow mode. Sizes of pixel, pinhole, and z-step were set to optimize resolution or to oversample in the case of images to be deconvolved. Deconvolution was performed using Huygens Professional (Scientific Volume Imaging, Hilversum, the Netherlands) using a theoretical point spread function, manual settings for background intensity, and default signal-to-noise ratio. Color balance, contrast, and brightness were adjusted with ImageJ.
3
APEX2-mediated Protein Localization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For initial testing of the APEX2 reaction, cells were grown on coverslips and transfected with the APEX2-lamin-B1 construct. The reaction was performed either with live cells, or after 1% PFA fixation as described above. The APEX2 reaction was confirmed by incubating with mouse-anti FLAG-M2 antibody (1:1,000; Sigma-Aldrich; F3165) overnight at 4°C and followed by incubation with streptavidin conjugated to Alexa 488 (1:200; Biolegend; 405235) and an anti-mouse secondary conjugated (1:1,000) to an Alexa fluorophore. Other primary antibodies used for immunofluorescence include rabbit anti-HNRNPA1 (1:2,500; ProteinTech; 11176-1-AP), rabbit anti-SRSF1 (1:2,500; ProteinTech; 12929-2-AP), rabbit anti-SRSF7 (1:2,500; Novus; NBP1-92382), rat anti-Nup153 (Abcam; ab81463), and rabbit anti-PCBP2 (Invitrogen; PAS-30116). For cell suspensions, all washing procedures were done by pelleting at 500 g × 5 m and resuspended in Prolong anti-fade gold for mounting and imaging. Imaging was done at room temperature using the Leica Application Suite software v2.7.3.9723, an SP5 confocal microscope (Leica), a 40×/1.3 NA oil objective, and Type F immersion liquid (Leica; 11513859).
4
Confocal Microscopy Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All samples were imaged using a Leica confocal SP8 microscope, with either a 40X 1.3NA (HC PL APO 40x/1.3 Oil CS2, for 20 mm sections) or a 20X 0.75NA (HC PL APO 20x/0.75 IMM CORR CS2, free working distance = 0.68 mm, for thick cleared slices) oil objective with type F immersion liquid (Leica, refractive index ne = 1.5180). After acquisition, stitched images were compensated for spectral overlap between channels using the Leica Channel Dye Separation module in the Leica LASX software. For single stained controls, UltraComp beads (Affymetrix) were incubated with fluorescently conjugated antibodies, mounted on slides, and imaged with the same microscope settings used in the image they were being used to compensate. In all figures, for visual clarity, thresholds were applied to the displayed channel intensities.
5
Visualizing Protein Dimerization in Live Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were maintained in 3.5 cm glass-bottom dishes (IWAKI, Tokyo, Japan), and the media was exchanged into DMEM without phenol red (Gibco) before observation. Cells were placed in a temperature chamber (TOKAI HIT, Fujinomiya, Japan) for live imaging. This temperature chamber kept the cells in a 37 °C, 5% CO2 environment.
The observation was carried out using a Leica TCS SP8 system (Leica, Tokyo, Japan) with a Leica DMi8 microscope equipped with a 63/NA-1.40 liquid immersion objective and Type F Immersion Liquid (Leica). The confocal microscope was controlled using the LAS X software (Leica, Tokyo, Japan). The Venus-fused protein was imaged and the iLID-SspB dimerization was activated using laser light (488 nm). RFP-fusion protein imaging was carried out using laser light (552 nm).
The observation was carried out using a Leica TCS SP8 system (Leica, Tokyo, Japan) with a Leica DMi8 microscope equipped with a 63/NA-1.40 liquid immersion objective and Type F Immersion Liquid (Leica). The confocal microscope was controlled using the LAS X software (Leica, Tokyo, Japan). The Venus-fused protein was imaged and the iLID-SspB dimerization was activated using laser light (488 nm). RFP-fusion protein imaging was carried out using laser light (552 nm).
6
Confocal Microscopy Imaging Protocol
7
Quantifying Protein Trafficking using LSCM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were imaged on an upright Leica DM 6000, TCS SP8 laser scanning confocal microscope with 405-nm, 488-nm, 552-nm and 638-nm lasers. The microscope uses two HyD detectors and three PMT detectors. The objective used was a ×63 HC PL APO CS2 oil objective with an NA of 1.40. Type F immersion liquid (Leica) was used for oil objectives. Images were 175.91 × 171.91 µm2, 1,024 × 1,024 pixels and 16 bits per pixel. For PTM quantification, HEK293T cells and human lung tissue were imaged at a single z plane and A549 cells were imaged with a z stack through the nucleus.
8
F-Actin Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For F-actin staining, cells were seeded as described for the uptake experiments. Cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at 37°C and permeabilized for 10 minutes with 0.1% Triton X-100. F-actin was stained with Alexa Fluor 555-conjugated phalloidin (Thermo Fisher Scientific). Cells were also stained with DAPI for the identification of nuclei. All steps during the staining process were conducted at room temperature in a dark room. After staining, cells were sandwiched between the glass coverslip and a glass slide and mounted with Fluoromount (Sigma-Aldrich Co.). Images were obtained with an Eclipse TE2000-U fluorescent microscope (Nikon Corporation, Tokyo, Japan) with a Plain Apo VC ×100 oil-immersion objective (Nikon Corporation) and Type F immersion liquid (Leica Microsystems).
9
Platelet Cytoskeletons Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After fixation in paraformaldehyde, platelets were cytospun, permeabilized with 0.1% Triton X-100 in PBS and incubated sequentially for 30 min with an anti-ß1-tubulin mAb (1:400, 1 μg/mL, Eurogentec, Liège, Belgium) followed by a secondary GAM-488 antibody (10 µg/mL) and an anti-GPIIbIIIa mAb (Alma.17–647, 10 µg/mL) in PBS containing 1% BSA. The cells were then embedded in Mowiol (Mountant, Permafluor, Thermo Fisher Scientific, UK) and examined under a confocal microscope (TCS SP8, Leica Microsystems, Rueil-Malmaison, France) equipped with an oil objective (Type F immersion liquid, ne23 = 1,5180, ve = 46, Leica Microsystems). Data were acquired with LASAF software, version 1.62 (Leica Microsystems). Mouse lungs were embedded in a cryogenic gel and serial longitudinal cryosections were stained and observed as previously reported13 (link).
10
Wide-Field Fluorescence Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wide-field microscopy was performed on a Leica DM6000 (Leica Microsystems, Wetzlar, Germany; filter cubes: DAPI (Ex: 360/40, Em: 470/40), FITC (Ex: 480/40; Em: 527/30), TRITC (Ex: 546/12; Em: 600/40), Cy5 (Ex: 620/60; Em: 700/75)) using an HCX PL APO 100x/1.40-0.70 OIL objective, a Leica EL6000 with an HXP 120W/45C Vis Hg light source, Type F immersion liquid (Leica Microsystems), a Leica DFC350 FX camera, and Las X 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!