microcapillary holder  (Eppendorf AG)

Journal: Journal of Biological Methods

Article Title: Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol

doi: 10.14440/jbm.2018.253

Figure Lengend Snippet: A. Injection and electroporation of pCMV-IRES-GFP into the ES at stage HH16. Light green triangle represents the injection microcapillary and dark green dots represent where DNA was injected. Black and red bars indicate the position of the −ve and +ve electrodes respectively in relation to the ES. B. Example of an embryo electroporated with IRES-GFP at stage HH16 then incubated for 24 h, strong GFP fluorescence can be seen in somites. C. Enlarged electroporated somites of the embryo in (B). D. Embryo electroporated with IRES-GFP at stage HH16 and incubated for 72 h, strong GFP fluorescence signals can be seen in the somites. E. Embryo electroporated with IRES-RFP at stage HH16 and incubated for 72 h, RFP strong fluorescence signals can be seen in the somites. F. Transverse section of the embryo in (E) after processing for double ISH showing RFP (red) expression in the transfected somite domain (arrows) and MyoD expression (blue) in both injected (right) and non-injected control sides (left). GFP in (B-D) was detected by Alexa Fluor 488 ηm and RFP in (E) by Alexa Fluor 565 ηm fluorescence filters. av: auditory vesicle; e: eye; ES: epithelial somites; fb: fore-brain; fl: fore-limb; h: heart; hb: hind-brain; hl: hind limb; mb: mid-brain; nt: neural tube; psm: presegmented mesoderm. Scale bars: 0.5 mm in (A and D), 1.5 mm in (B), 0.4 mm (C), 0.3 mm in (E) and 0.1 mm in (F).

Article Snippet: Fill in the microcapillary with DNA/Fast Green mix or fluorescent CMO using Eppendorf microloader fine tip and then mount the microcapillary onto the microcapillary holder.

Techniques: Injection, Electroporation, Incubation, Fluorescence, Expressing, Transfection

Journal: Journal of Biological Methods

Article Title: Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol

doi: 10.14440/jbm.2018.253

Figure Lengend Snippet: Troubleshooting.

Article Snippet: Fill in the microcapillary with DNA/Fast Green mix or fluorescent CMO using Eppendorf microloader fine tip and then mount the microcapillary onto the microcapillary holder.

Techniques: Incubation, Electroporation, Injection, Concentration Assay, Expressing, Fluorescence, Microscopy

Journal: Journal of Biological Methods

Article Title: Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol

doi: 10.14440/jbm.2018.253

Figure Lengend Snippet: A. Injection and electroporation of pCMV-IRES-GFP into the ES at stage HH16. Light green triangle represents the injection microcapillary and dark green dots represent where DNA was injected. Black and red bars indicate the position of the −ve and +ve electrodes respectively in relation to the ES. B. Example of an embryo electroporated with IRES-GFP at stage HH16 then incubated for 24 h, strong GFP fluorescence can be seen in somites. C. Enlarged electroporated somites of the embryo in (B). D. Embryo electroporated with IRES-GFP at stage HH16 and incubated for 72 h, strong GFP fluorescence signals can be seen in the somites. E. Embryo electroporated with IRES-RFP at stage HH16 and incubated for 72 h, RFP strong fluorescence signals can be seen in the somites. F. Transverse section of the embryo in (E) after processing for double ISH showing RFP (red) expression in the transfected somite domain (arrows) and MyoD expression (blue) in both injected (right) and non-injected control sides (left). GFP in (B-D) was detected by Alexa Fluor 488 ηm and RFP in (E) by Alexa Fluor 565 ηm fluorescence filters. av: auditory vesicle; e: eye; ES: epithelial somites; fb: fore-brain; fl: fore-limb; h: heart; hb: hind-brain; hl: hind limb; mb: mid-brain; nt: neural tube; psm: presegmented mesoderm. Scale bars: 0.5 mm in (A and D), 1.5 mm in (B), 0.4 mm (C), 0.3 mm in (E) and 0.1 mm in (F).

Article Snippet: For setting up the electroporation station ( ): (1) Obtain a commercially available galvanized iron sheet with approximate measurements of 80 cm length, 40 cm width, and 3 mm thickness, and then place the iron sheet in the middle of the electroporation station; (2) Place the stereomicroscope with its associated reflected light units in the middle of the iron sheet; (3) Place a manual micromanipulator (loaded on a mounting magnetic base) on each side of the stereomicroscope, one micromanipulator should hold the injection microcapillary holder and another should carry the electrodes’ holder; switch the magnetic controls of the mounting bases “on” to fix them firmly in place on the iron sheet; (4) Place the Eppendorf microinjector ( ) on one side of the electroporation station and the electroporator (connected to the current amplifier) ( ) on the other side; and (5) Connect the injection microcapillary to the microinjector using the provided tubing and the electrodes to the electroporator using the provided electric wires.

Techniques: Injection, Electroporation, Incubation, Fluorescence, Expressing, Transfection

Journal: Journal of Biological Methods

Article Title: Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol

doi: 10.14440/jbm.2018.253

Figure Lengend Snippet: Troubleshooting.

Article Snippet: For setting up the electroporation station ( ): (1) Obtain a commercially available galvanized iron sheet with approximate measurements of 80 cm length, 40 cm width, and 3 mm thickness, and then place the iron sheet in the middle of the electroporation station; (2) Place the stereomicroscope with its associated reflected light units in the middle of the iron sheet; (3) Place a manual micromanipulator (loaded on a mounting magnetic base) on each side of the stereomicroscope, one micromanipulator should hold the injection microcapillary holder and another should carry the electrodes’ holder; switch the magnetic controls of the mounting bases “on” to fix them firmly in place on the iron sheet; (4) Place the Eppendorf microinjector ( ) on one side of the electroporation station and the electroporator (connected to the current amplifier) ( ) on the other side; and (5) Connect the injection microcapillary to the microinjector using the provided tubing and the electrodes to the electroporator using the provided electric wires.

Techniques: Incubation, Electroporation, Injection, Concentration Assay, Expressing, Fluorescence, Microscopy

Journal: Nature protocols

Article Title: Generation of Expandable, Multipotent Induced Cardiac Progenitor Cells from Mouse Fibroblasts and Potency Testing in ex vivo Embryos

doi: 10.1038/nprot.2017.021

Figure Lengend Snippet: (a) Complete frozen Embryo Dissection Medium (right) and frozen Incomplete Whole Embryo Culture Medium (without serum) (left). Upon thawing, the Incomplete Whole Embryo Culture Medium will turn pink. (b) Comparison of non-hemolyzed (right) and hemolyzed (left) rat serum. (c) The mouth aspirator assembly (center) is bought from Sigma. Its major useful feature is the microcapillary holder (1). The holder can serve as an adaptor for the microflame (left), which consists of the adaptor minus the gasket, a piece of latex tubing and a cut-off 19-g hypodermic needle. A piece of wider-gauge latex tubing is then secured over the adaptor and connected to the gas outlet. The round mouthpiece (2) is replaced by a flat mouthpiece that is inserted into a disposable filter (0.22 or 0.45 μm) using parafilm to create a tight seal (not shown). The filter is then secured onto a longer piece of latex tubing followed by the microcapillary and its gasket, which securely holds an injection pipette. (d) The miciroflame (left) will be secured onto a clamp (right) and connected to the gas outlet (not shown). (e) The microflame is ignited. (f, g) A piece of glass microtubing is briefly softened at its center (f) and, when red hot, removed from the flame and pulled horizontally (g). (h, i) The pulled microcapillary is loaded onto an electrode puller, the trough filament of which is heated (h), and the glass is then pulled, creating two tapered glass capillaries (i, only one is shown). (j) The glass capillaries are cut to the right diameter (20 μm) on a microforge, in the following steps: (1) The glass bead (black) stuck to the platinum wire is made to approach the glass capillary at the desired diameter (measured with an eyepiece reticle). (2) The bead is placed onto the glass microcapillary and the heat is momentarily turned on. (3) Turning the filament off results in contraction of the glass bead, bringing with it the distal portion of the fused glass filament. (4) The end of the injection pipette is fire-polished by aligning the glass bead to its opening and briefly turning on the heat. (k) Once the capillaries are microforged, they can be stored in a Nalgene box into which a piece of flexible Styrofoam has been glued (Glue-Stick); the Styrofoam is scored with a razor blade, and the capillaries inserted up to three deep into the scored Styrofoam.

Article Snippet: Sigma supplies mouth aspirator tube assemblies that come with a round mouth aspirator, about a foot of latex tubing, and a microcapillary holder.

Techniques: Dissection, Embryo Culture, Injection, Transferring