Bacteria were grown until OD620 = 0.15 and competence was induced as described above. 10 ml of culture were harvested by centrifugation for 5 min at 2,700 g, 4°C. The pellet was resuspended in 5 ml PBS containing 0.5% BSA. 10 µl of this suspension was placed on a coverslip (VWR) and air-dried. Bacteria were fixed in PBS containing 4% paraformaldehyde for 30 min and coverslips were washed three times with PBS containing 0.5% BSA. Samples were labeled with primary antibodies to ComGC, ComGD, ComGF or ComGG, diluted at 1:300, and secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Life technologies) or secondary rabbit anti-goat antibody conjugated with Alexa Fluor 488 (Abcam) for 1 h. Flag-tagged proteins were labeled with anti-Flag M2 monoclonal antibody (Sigma) diluted 1:300 and secondary goat anti-mouse antibody conjugated with Alexa Fluor 594 for 1h. coverslips were mounted onto microscope slides with Vectashield (Vector Laboratories). Samples were examined using a DV Elite microscope (Applied Precision) equipped with a scientific complementary metal-oxide-semiconductor (sCMOS) camera. Images were acquired using Softworx (Applied Precision) and analyzed in ImageJ (Schneider et al., 2012 (link)).
Coverslip
Manufactured by Avantor
Sourced in United States, United Kingdom, Canada, Germany
Coverslips are transparent thin glass or plastic sheets used to cover and protect specimens in various microscopy applications. They provide a flat, even surface to place samples on and facilitate clear observation and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (7 mentions)
Fluoromount g, by Southern Biotech (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Click it reaction kit, by Thermo Fisher Scientific (4 mentions)
Normal goat serum (ngs), by GeneTex (4 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Microscope slide, by Avantor (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 510 meta confocal microscope, by Zeiss (3 mentions)
Carbo free blocking solution, by Vector Laboratories (2 mentions)
Dabco, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Zen black software, by Zeiss (2 mentions)
Elyra ps1 system, by Zeiss (2 mentions)
Blocking powder, by PerkinElmer (2 mentions)
Antigen unmasking solution, by Vector Laboratories (2 mentions)
Laminin, by Merck Group (2 mentions)
67 protocols using coverslip
1
Immunofluorescent Localization of Competence Proteins
2
Cell Migration and Invasion Assay
3
Immunofluorescent Staining of Muscle Cells
4
Imaging and Quantifying Microvilli Dynamics
5
Screening for Bacillus Crystal Proteins
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Isolates were grown on T3 agar for 3 days at 30°C to promote sporulation and crystal protein production. After completed incubation, one colony was resuspended in 10 µL of PBS on a microscope slide and covered with a cover slip (VWR international, Pennsylvania, USA) and screened within 10 minutes for the presence of crystal proteins using a phase-contrast microscope (Olympus BX51, Olympus, Tokyo, Japan). Crystal proteins were differentiated by shape (bipyramidal, cuboidal, and circular), size (smaller than a vegetative cell and endospore of Bacillus), and color (darker than Bacillus endospore). Initial screening results were classified as “positive,” “negative,” or “inconclusive.” Screening was repeated three times by two individuals to assess the production of crystal proteins.
6
Confocal Microscopy of Mayonnaise Microstructure
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Mayonnaise and analog microstructure were analyzed with a Nikon C2 confocal laser scanning microscope (CLSM) (Nikon, Mississauga, ON, Canada) using a 543 nm laser with a 60× Plan-Apochromat VC (numerical aperture 1.4) oil immersion objective lens and five times digital zoom. The oil phase was stained using Nile red dye (0.01 wt.%). A drop of emulsion was placed on a microscope slide (Fisher Scientific, Nepean, ON, Canada) with a glass rod, covered with a coverslip (VWR International, Edmonton, AB, Canada) and observed under the CLSM.
7
Immunofluorescent Staining of Muscle Cells
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TA or Plantaris muscle sections, SMs or cytospun MuSCs were fixed for 10
min in 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 (Sigma)/PBS for
10 min at room temperature (RT), rinsed with wash buffer (0.05% Triton-X
100/PBS), treated with blocking buffer (10% Normal Goat Serum (Genetex) and
1× carbo-free blocking solution(Vector)) for 1 h, prior to incubation
with primary antibodies (seeSupplementary Table ) diluted in blocking buffer overnight at 4
°C. Samples were then washed with wash buffer and incubated with
appropriate Alexa-Fluor-conjugated secondary antibodies (1:1,000, Life
Technologies) and in blocking buffer for 1 h at RT. After wash and incubation
with 4′,6-diamidino-2-phenylindole (DAPI) at1 μg/ml for 5 min,
samples were mounted with Fluoromount-G (SouthernBiotech) and coverslip (VWR).
For EdU detection, the Click-iT reaction kit (Life Technologies) was used prior
to incubation in DAPI according to manufacturer’s recommendations.
min in 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 (Sigma)/PBS for
10 min at room temperature (RT), rinsed with wash buffer (0.05% Triton-X
100/PBS), treated with blocking buffer (10% Normal Goat Serum (Genetex) and
1× carbo-free blocking solution(Vector)) for 1 h, prior to incubation
with primary antibodies (see
°C. Samples were then washed with wash buffer and incubated with
appropriate Alexa-Fluor-conjugated secondary antibodies (1:1,000, Life
Technologies) and in blocking buffer for 1 h at RT. After wash and incubation
with 4′,6-diamidino-2-phenylindole (DAPI) at1 μg/ml for 5 min,
samples were mounted with Fluoromount-G (SouthernBiotech) and coverslip (VWR).
For EdU detection, the Click-iT reaction kit (Life Technologies) was used prior
to incubation in DAPI according to manufacturer’s recommendations.
8
Immunofluorescent Staining of Myelin Proteins
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Human sections were first fixed in paraformaldehyde (PFA) 4% for 5 min. For myelin protein staining, tissues were pre-incubated in absolute ethanol at −20 °C for 20 min. Tissues were blocked in PBS, 5–10% Normal Goat Serum (50-062Z; Thermo Fisher Scientific), 0.2–0.4% Triton X-100 (Sigma), then incubated with primary antibodies in blocking solution overnight at room temperature, washed in PBS, and incubated with secondary antibodies for 1–3 h at RT in the dark. When applicable, tissues were immersed for 30 s in Hoechst solution (10 μg/ml, Euromedex). They were finally mounted under coverslip (VWR) with Fluoromount (Southern Biotech).
9
Laser-induced Dendrite Regeneration Imaging
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Three-day-old larvae reared at 25°C were removed from their food cap and gently washed in water. They were then mounted on a glass microscope slide with a pad of dried 3% agar (for stability) and immobilized with a coverslip (40 × 22 mm; VWR) held down by tape. Larvae had the comb/main dendrite of one ddaE neuron severed with a MicroPoint pulsed UV laser (Oxford Instruments/Andor, USA) and were imaged on either a Zeiss confocal LSM700, LSM800, or widefield microscope. Postinjury and -imaging, the larvae were returned to a small piece of fly media in a cap and kept at 25°C for 24 h. Larvae were remounted and reimaged using a Zeiss LSM800 confocal or widefield microscope. Regeneration was quantified as (total branch points at 24 h postinjury) – (total branch points on remaining dendrite at time of injury). For experiments in the heterozygous γTub23C mutant background, a red cell shape marker was used, while other experiments included a green cell shape marker. When using the red cell shape marker, some images of injured neurons were too dim to accurately quantify and were excluded from analysis.
10
Emulsions Microscopy Characterization
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The emulsions were imaged and characterized by bright field and fluorescence microscopy within a day after their preparation.
The microscopy samples were prepared by carefully pipetting the emulsion droplets in a custom-made cell consisting of two glass slides (Thermo Scientific Menzel Gla ¨ser microscope slide 76 Â 26 mm 2 ) glued on top of each other, where a hole of 1.5 cm in diameter had been cut out. The cell was sealed with a cover glass (VWR cover slip 22 Â 22 mm 2 , #1.5 thick).
The obtained emulsion type (o/w or w/o) was observed with a Nikon Eclipse TE2000-U equipped with a Nikon D-Eclipse C1 laser scanning confocal microscope system. A 488 nm Ar laser was used for the excitation of the BODIPY dye in the oil phase. In the case of the co-existence of w/o and o/w, the dominating emulsion type has been estimated via visual inspection comparing the numbers of bright and dark pixels of several images.
Bright field images were collected with either a Nikon Eclipse Ti-E microscope with a Hamamatsu C11440 ORCA-Flash4.0 digital camera (2048 Â 2044 px images) or a Nikon Eclipse Ti-U microscope with an Imaging Source DFK 33UX249 (1920 Â 1200 px images).
The microscopy samples were prepared by carefully pipetting the emulsion droplets in a custom-made cell consisting of two glass slides (Thermo Scientific Menzel Gla ¨ser microscope slide 76 Â 26 mm 2 ) glued on top of each other, where a hole of 1.5 cm in diameter had been cut out. The cell was sealed with a cover glass (VWR cover slip 22 Â 22 mm 2 , #1.5 thick).
The obtained emulsion type (o/w or w/o) was observed with a Nikon Eclipse TE2000-U equipped with a Nikon D-Eclipse C1 laser scanning confocal microscope system. A 488 nm Ar laser was used for the excitation of the BODIPY dye in the oil phase. In the case of the co-existence of w/o and o/w, the dominating emulsion type has been estimated via visual inspection comparing the numbers of bright and dark pixels of several images.
Bright field images were collected with either a Nikon Eclipse Ti-E microscope with a Hamamatsu C11440 ORCA-Flash4.0 digital camera (2048 Â 2044 px images) or a Nikon Eclipse Ti-U microscope with an Imaging Source DFK 33UX249 (1920 Â 1200 px images).
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