We carried out three different stab wound injury paradigms: nostril, skull, and small skull injuries (Figures 1 and 6). Fish were anesthetized with buffered 0.02% MS-222 for 45 s to a minute and then placed in a Tricaine-soaked sponge. With the visual aid of a dissecting microscope, injuries were performed in both telencephalic hemispheres. The nostril injury [37 (link)] was performed using a 100 × 0.9 mm glass capillary needle (KG01, A. Hartenstein). Capillaries were pulled on a Narishige Puller (model PC-10) using a “One-stage” pull setting at a heater level of 63.5 °C. The final dimensions of the capillaries were 5 mm in length and 0.1 mm in diameter. For the skull injury, a micro-knife (Fine Science Tools) was inserted vertically through the skull into the medial region of the telencephalon. To perform the small skull injury, the skull was thinned above the telencephalon area using a micro-driller (Foredom) and the glass capillary (identical to that used for the nostril injury) was inserted vertically through the skull and brain parenchyma. After the injury, fish were placed in fish water with oxygenation to assure complete recovery from the anesthesia.
Micro knife
Manufactured by Fine Science Tools
The Micro-knife is a precision instrument designed for delicate cutting tasks in laboratory settings. It features a fine, sharp blade that allows for controlled and accurate cuts on small samples or materials. The Micro-knife is a versatile tool suitable for a range of fine dissection, trimming, and preparation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Halocarbon oil, by Merck Group (1 mentions)
Microinjector, by Eppendorf (1 mentions)
Zymosan, by Merck Group (1 mentions)
Fast green dye, by Merck Group (1 mentions)
Nanoject 3 injector, by Drummond (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
Neurobasal media, by Thermo Fisher Scientific (1 mentions)
7 protocols using micro knife
1
Three Telencephalic Injury Paradigms in Fish
2
Pupal Wing Imaging and Time-lapse Analysis
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Prepupae of indicated genotypes were raised and collected at room temperature, then shifted to 29 °C until staged appropriately (late inflation stage, roughly 18 h AP equivalent at 25 °C). Pupae were retrieved, briefly rinsed in water, dried on a Kimwipe, then positioned on a piece of double-sided tape (right wing facing up). Windows were carefully dissected into the pupal cases in the region of the wing using a microknife (cat# 10316–14; Fine Science Tools) essentially as described (51 (link)), avoiding damage to the underlying tissue. A tiny drop of halocarbon oil (Sigma Aldrich) was applied to the exposed pupal wing with a disposable pipet tip to prevent tissue desiccation during imaging. The pupae, adhering to strips of double-sided tape cut with a disposable scalpel, were then placed oil-side down onto a 24 × 50 mm coverslip. After 5–6 pupae were collected onto the coverslip, wings were time-lapse imaged on a Leica SP8 STED confocal microscope by taking optical anteroposterior cross sections of each wing every 4–5 min using the xzyt-function. The resulting time lapse images were processed into AVI-format videos using Imaris v.9.1.2 (Bitplane/Oxford Instruments).
3
Zymosan Microinjection into Fish Brain
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Fish were anaesthetized in 0.02% MS-222 and a small hole, using a micro-knife (Fine Science Tools), was made into the skull (above the telencephalic ventricle). A glass capillary loaded with 10 mg/mL Zymosan or artificial cerebrospinal fluid with Fast Green dye to visualize the injection site (0.3 mg/mL; Sigma) was inserted into the hole and ~1 μL of solution was injected at a pressure of 150 hPa using a microinjector (Eppendorf, Hamburg, Germany). Artificial cerebrospinal fluid was used as a control for the ventricular injections since its composition closely matches the electrolyte concentrations of cerebrospinal fluid (Figure 2).
4
Single-cell Sequencing of Tarsus Segments
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The following procedure was used to collect known-age first tarsal segments for single-cell sequencing. White P1 prepupae (0 to 1 h APF) from the DGRP line RAL-517 [175 (link)] were collected and sexed based on the presence of testes. Males were transferred to a folded kimwipe, wet with 500 μl of water, and held inside of a petri dish in an incubator maintained at 25°C on a 12:12 cycle. Pupae were removed from puparia using forceps and placed on top of a water-soaked kimwipe, 1 h before the desired age was reached (e.g., 23 h after collection for the 24 h sample). When the desired age was reached, pupae were placed ventral side up on tape. The base of the abdomen was pierced to release some of the fluid pressure and the foreleg removed at the tibia/tarsal joint. The dissected leg was then placed on tape, covered in a drop of 1X Dulbecco’s PBS (DPBS, Sigma, D8537), and a Micro Knife (Fine Science Tools, 10318–14) was used to sever at approximately the midpoint of the second tarsal segment. The first tarsal segment was then eased out of the pupal cuticle and transferred to a glass well on ice containing 100 μl of 1X DPBS using a BSA-coated 10-μl tip.
5
Axolotl Telencephalon Injury Protocol
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To access the telencephalon of anesthetized axolotls, two rectangular scalp skin flaps followed by cranial flaps were created dorsally with spring scissors. Incisions outlining a 0.8 mm X 1 mm rectangular injury site were made in the left dorsal pallium with a microknife (Fine Science Tools, Foster City, CA) secured onto a micromanipulator. The caudal-medial corner of the rectangular injury site is defined as 1 mm lateral from and 2 mm rostral from the choroid plexus. The injury site tissue was removed using No.5 forceps. After the injury, the cranial flaps were replaced, and the skins flaps were secured with non-absorbable silk sutures (Myco Medical, Cary, NC). In sham injury, the telencephalon was similarly exposed but the dorsal pallium was left intact.
6
Viral Transduction of Somatosensory Cortex
7
Time-lapse Imaging of Tissue Slices
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Embryos electroporated with pCAG>nls-Cas9-nls-2a-Cit-HH-Sox10.gRNAf+e-HDV, pCAG>H2B-RFP and FoxD3-NC2>Cerulean plasmids were incubated at 37°C, harvested after 24 h, and screened for the expression of nuclear RFP, which was co-electroporated as a transfection control. Embryos that were poorly transfected were discarded, and the rest were processed as follows: transverse cuts spanning two somites were made through the trunk region using a micro-knife (Fine Science Tools). The slices were washed in Ringer's solution and transferred into a fluorodish containing pre-warmed Neuro-basal media (Gibco) supplemented with glutamine and PenStrep. The slices were positioned under custom-made nylon grids and given 15 min to stabilize, after which the whole fluorodish was transferred into the incubation chamber of a Zeiss LSM 800 microscope for time-lapse imaging. Control embryos were processed in the same way as the mutant embryos.
For imaging the tissue slices, one-photon laser excitation was used at wavelengths of 405, 488 and 561 nm. For these experiments, the 20×/0.8 NA M27 objective was used and z-stacks comprising 25 slices spanning 38.4 µm were collected every 10 min for 11 h. The images were imported into Imaris to generate movies.
For imaging the tissue slices, one-photon laser excitation was used at wavelengths of 405, 488 and 561 nm. For these experiments, the 20×/0.8 NA M27 objective was used and z-stacks comprising 25 slices spanning 38.4 µm were collected every 10 min for 11 h. The images were imported into Imaris to generate movies.
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