Protoplasts were transfected with 10 or 20 μg pCDB-Cas9-GFP-PDS or pKAR6 in at least three independent experiments. The pKAR6 vector (Robert Blanvillain, unpublished data) (Thomson et al., 2011 (link); Supplementary Table 2 ) expresses GFP under a 35S promotor and was used as a positive control (PC) for protoplast transfection. Protoplasts transfected without vector (Negative Control 1; NC1) and protoplasts without the addition of both PEG and vector (Negative Control 2; NC2), were used as negative controls. After transfection, 1 mL of 0.5 M mannitol was added to the protoplast pellet and the protoplast suspension was transferred into a 6-well plate and cultured in the dark at 23 ± 2°C on an orbital shaker (30 rpm, 20 h). Next, the protoplast suspension was transferred to an Eppendorf tube, centrifuged for 5 min at 80 g in a swing out centrifuge and supernatant was removed. Twenty μL of the protoplast suspension was transferred to a Bürker chamber and analyzed with a Zeiss AxioImager M2 fluorescence microscope equipped with an Axiocam MRm camera and ZEN software and magnification 200 × (Carl Zeiss MicroImaging, Belgium). Transfection efficiencies were calculated as the ratio of the number of GFP expressing protoplasts (GFP Zeiss filter set 10 (excitation 489 nm, emission 509 nm) to the total number of living protoplasts (based on round shape under bright field microscopy).
Axioimager m2 fluorescence
Manufactured by Zeiss
Sourced in Germany
The Zeiss AxioImager M2 is a fluorescence microscope designed for high-resolution imaging. It features a motorized nosepiece, automated filters, and advanced illumination system to facilitate detailed analysis of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrm, by Zeiss (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Filter set 49, by Zeiss (1 mentions)
Sybr green 1 sbi, by Thermo Fisher Scientific (1 mentions)
Filter set 38, by Zeiss (1 mentions)
Filter set 20, by Zeiss (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 protocols using axioimager m2 fluorescence
1
Transient Transformation of Plant Protoplasts
2
Microscopic imaging of endolithic microbiome
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Small pieces of gypsum, colonized by pigmented endoliths were scraped and suspended in double-distilled water. The suspension was stained with SYBR Green I (SBI) (Molecular Probes), which is a fluorochrome specifically used for the staining of nucleic acids. Observations were made first in differential interference contrast (DIC) using a Zeiss AXIO Imager M2 fluorescence microscope (Carl Zeiss, Jena, Germany) plus an Apochrome x60, n = 1.4 Zeiss oil-immersion objective. A CCD Axiocam HRc Rev. 2 camera and AXIOVISION 4.7 software (Carl Zeiss, Oberkochen, Germany) were used to capture and record the DIC images. Images were acquired using a Multichannel Image Acquisition system, employing an eGFP filter set (Zeiss Filter Set 38; Ex/Em: 450–490/500–550 nm), a DAPI filter set (Zeiss Filter Set 49; Ex/Em: 365/420–470 nm), and a Rhodamine filter set (Zeiss Filter Set 20; Ex/Em: 540–552/567–647 nm).
3
Fluorescence Imaging of Leaf Sections
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After incubation, leaf sections were transferred to a microscope slide with the adaxial side facing up and covered with 10–50 μl Aqua Poly/Mount (Polyscience Inc., Warrington PA, USA) medium to ensure a good coverage of the sample. We then carefully added a cover slip which we fixed with strips of adhesive tape to all sides. From each leaf section, we typically took 10 micrographs at random positions using an Axio Imager.M2 fluorescence microscope (Carl Zeiss AG, Oberkochem, Germany) equipped with EC Plan-Neofluar10x/0.3, 20x/0.5 and 40x/0.6 (Zeiss) objectives and an AxioCAM MRn monochrome camera. Image sizes were 895.3 μm × 670.8 μm, 447.6 μm × 335.4 μm and 223.8 μm × 167.7 μm for the 10×, 20× and 40× objective, respectively, but in few instances smaller, when out-of-focus areas had to be cropped. For the fluorescence images, we used a GFP filter cube (exciter: 470; emitter: 525/50; beam splitter: 495) and a rhodamine filter cube (exciter: 546/12; emitter: 607/80; beam splitter 560). We also took phase-contrast images of all samples to visualize the leaf surface structure. To account for the topography of the leaf surface, we took all images as 3D ‘z-stacks’, i.e. several shots of the same area at different planes of focus. These were saved in the native Zeiss .zvi format.
4
Immunofluorescence Staining and Mitochondria Analysis
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Tissues were isolated and fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. Fixed tissues were dehydrated by incubation in a series ethanol gradient and Xylene, then embedded in paraffin wax for sectioning at the thickness of 5 μm. Slices obtained were rehydrated and boiled in Sodium Citrate solution at 120°C for 20 min for antigen retrieval. Slices were washed in PBS once before the blocking step. Cells cultured on the coverslip were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. Subsequently, coverslips were washed in PBS three times before blocking.
The brain slices or coverslips were incubated for 1 h in blocking solution containing 5% bovine serum albumin (BSA, Sigma) and 0.1% Triton X-100 (Sigma) in PBS. They were then incubated with primary antibody overnight at 4°C, followed by washing three times in PBS before incubation with fluorophore-conjugated secondary antibodies. After secondary antibody incubation, they were stained with DAPI, washed three times and mounted with Prolong Gold Mounting Medium (Thermo) for imaging. Images were captured using a Zeiss Axio Imager M2 fluorescence microscope. Image analysis was done using ImageJ. The mitochondria morphology was analyzed by ImageJ plugin MiNA (Valente et al., 2017 (link)).
The brain slices or coverslips were incubated for 1 h in blocking solution containing 5% bovine serum albumin (BSA, Sigma) and 0.1% Triton X-100 (Sigma) in PBS. They were then incubated with primary antibody overnight at 4°C, followed by washing three times in PBS before incubation with fluorophore-conjugated secondary antibodies. After secondary antibody incubation, they were stained with DAPI, washed three times and mounted with Prolong Gold Mounting Medium (Thermo) for imaging. Images were captured using a Zeiss Axio Imager M2 fluorescence microscope. Image analysis was done using ImageJ. The mitochondria morphology was analyzed by ImageJ plugin MiNA (Valente et al., 2017 (link)).
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