To stain small numbers of neurons (1–20) with processes in the anterior optic tubercle (AOTU), we used extracellular iontophoretical dye injections. Intracellular recording pipettes with resistances between 100 and 300 MΩ in the tissue were fabricated as described above. Electrode tips were filled with 4% Neurobiotin (Vector Laboratories Burlingame, USA) in 1 M KCl and backed with 1–2.5 M KCl. After removing the neural sheath, electrodes were frontally inserted into the AOTU using a micromanipulator (Leica Microsystems, Wetzlar, Germany). To eject the tracer, and to create an electroporating electrical field [31 (link)], we applied rectangular current pulses of 10 nA amplitude with a frequency of 1 Hz and a duty cycle of 50% for 15 to 45 minutes, using a custom built amplifier.
Micromanipulator
Manufactured by Leica
Sourced in Germany, Canada
The Leica Micromanipulator is a precision instrument designed for accurate and controlled manipulation of microscopic samples. It provides precise three-dimensional movement of a microscopic probe or tool within a microscope's field of view, allowing for delicate and intricate procedures on small-scale specimens.
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Lab products found in correlation
Inverted microscope, by Leica (3 mentions)
Dmi3000b microscope, by Leica (3 mentions)
Dmi3000 b inverted scope, by Leica (2 mentions)
Picospitzer 3, by Leica (2 mentions)
P 97 micropipette puller, by Sutter Instruments (2 mentions)
Femtojet, by Eppendorf (2 mentions)
Neurobiotin, by Vector Laboratories (1 mentions)
Guillard s f 2 medium, by Merck Group (1 mentions)
Tcm 199, by Thermo Fisher Scientific (1 mentions)
Hanks salts, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Microtome, by Zeiss (1 mentions)
M16 medium, by Merck Group (1 mentions)
M2 medium, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Mmessage mmachine t3 kit, by Thermo Fisher Scientific (1 mentions)
Needle puller, by Narishige (1 mentions)
Fluorescence stereomicroscope, by Leica (1 mentions)
P 2000 laser puller, by Sutter Instruments (1 mentions)
Embryomax m2 medium, by Merck Group (1 mentions)
21 protocols using micromanipulator
1
Selective Neuron Staining in Anterior Optic Tubercle
2
Morpholino Knockdown of Ambra1a/b
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MO (Gene Tools) treatment was performed with MOs against the ATG translation initiation sites of either ambra1a or ambra1b transcripts (MO-ambra1a-ATG and MO-ambra1b-ATG) and with splice-blocking MOs designed at the exon 3-intron 3 junction sequence of both genes (MO-ambra1a-splice and MO-ambra1b-splice). The designed splice-blocking MOs cause the skipping of exon 3, thus altering the translation reading frame of exon 4 with introduction of a premature stop codon, and the resulting proteins lack all known binding domains (Fig. S1 ). As controls, we used five-nucleotide-mismatched control MOs (MO-ambra1a-5m and MO-ambra1b-5m). All MOs were previously described and validated [14] (link), however lower MOs dosages were used in this work in order to reduce embryo mortality. Specifically, for each MO, 10.3 ng were injected in the yolk of 1-cell stage embryos, whereas the dosage was halved in the co-injection experiments. Injections were performed under a dissecting microscope using a microinjector attached to a micromanipulator (Leica Microsystems). MOs-injected embryos were then incubated in 1x fish water solution at 28.5 °C up to the desired stages of development.
3
Isolation and Culture of Epiphytic Diatom
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S. unipunctata was isolated from samples of P. oceanica leaves collected in the field from the meadows located in Lacco Ameno (Island of Ischia, Gulf of Naples, Italy). Once in the laboratory, the lamina of each leaf was rinsed with filtered seawater (FSW) and then gently scraped with a glass slide in order to collect the epiphytic communities. The diatoms of interest were isolated under inverted microscope (Leica Microsystems), through sequential transfer of single cells, by means of a micromanipulator (Leica Microsystems). The isolated diatom was deployed in multi-well plates filled with sterile seawater in order to obtain axenic cultures. The monoclonal strains thus obtained were gently renovated under a laminar flow hood and cultured in 12-multiwell plates with Guillard’s f/2 medium (Sigma-Aldrich, Milan, Italy) and kept in a thermostatic chamber at 18 °C, with a 12 h:12 h light:dark photoperiod. Light was provided by Silvania GroLux (Osram Sylvania Inc., Wilmington, Massachusetts, USA) at 140 μE∙m−2∙s−1 irradiance.
4
Blastocyst Biopsy Technique
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Blastocyst biopsy was performed as described by de Sousa et al. [30 (link)] with minor modifications. Briefly, blastocysts were cut using a micromanipulator (Leica Microsystems, Wetzlar, Germany) and a stainless-steel blade at an angle of 30° (Bio-Cut-Blades Feather; Feather Safety Razor Co., Osaka, Japan). Embryos were micromanipulated on a 90 × 15 mm Petri dish (AS ONE Corporation, Osaka, Japan) containing 200 μL holding medium consisting of TCM–199 and Hanks’ salts (Invitrogen, Carlsbad, CA) supplemented with 0.3% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO).
5
Transient Protein Expression in Zebrafish Embryos
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One- to two-cells stage embryos were microinjected into the yolk mass with 10 nl of a solution containing 30 ng per μl of pCS2 + hAMBRA1-RFP-sspB (or pCS2 + RFP-sspB as a control) and 15 ng per μl of pCS2 + Venus-iLID-ActA in ×1 Danieau’s buffer. Injections were performed under a dissecting microscope using a microinjector attached to a micromanipulator (Leica Microsystems, Milan, Italy). Injected embryos were raised to the desired stages for in vivo imaging or collected for further analyses.
6
Visualizing Dnmt1 Isoforms in Mouse Zygotes
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Mouse zygotes were collected from superovulated BCF1 (C57BL/6 × CBA/CA) females as described previously (28 ). Briefly, female BCF1 mice at 5 weeks of age were injected with 5 IU of pregnant mare serum gonadotrophin (PMSF), followed by 5 IU of human chorionic gonadotropins (hCG) 48 h apart, and mated with male mice. Successful mating was determined the following morning by detection of a vaginal plug. Mouse zygotes were transferred to M2 medium (Sigma) containing 0.1% (w/v) hyaluronidase to remove cumulus cells and cultured in M16 medium (Sigma) at 37°C, 5% CO2 in air (29 (link)).
Microinjection was performed 24 h after human chorionic gonadotropin injection (30 (link)). To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min. Linearized GFP-Dnmt1sc− or GFP-Dnmt1oc− expression construct (4–20 ng/μl) was microinjected into the male pronuclei of mouse zygotes using an inverted microscope equipped with a micromanipulator (Leica). The injected zygotes were then cultured in M16 media for 48 h. The resulting four- or eight-cell stage embryos were collected, fixed using 4% formaldehyde and mounted with DAPI (Vector). The localization of Dnmt1 isoforms was evaluated by detecting GFP signals under a fluorescence microscope equipped with a microtome (Zeiss).
Microinjection was performed 24 h after human chorionic gonadotropin injection (30 (link)). To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min. Linearized GFP-Dnmt1sc− or GFP-Dnmt1oc− expression construct (4–20 ng/μl) was microinjected into the male pronuclei of mouse zygotes using an inverted microscope equipped with a micromanipulator (Leica). The injected zygotes were then cultured in M16 media for 48 h. The resulting four- or eight-cell stage embryos were collected, fixed using 4% formaldehyde and mounted with DAPI (Vector). The localization of Dnmt1 isoforms was evaluated by detecting GFP signals under a fluorescence microscope equipped with a microtome (Zeiss).
7
Overexpression and Depletion of Embryonic Factors
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Zygotes from superovulated and mated F1 females were isolated 20 h post-hCG injection. To overexpress p54nrb or CARM1-WT embryos were microinjected with a synthetic mRNA (50 ng μl−1, 100 ng μl−1 or 400 ng μl−1) into the cytoplasm between 24 and 27 h after hCG injection, using an Eppendorf micromanipulator on a Leica inverted microscope. As marker of injection either Gap43-GFP or Gap43-RFP mRNA (200 ng μl−1) were used. Embryos were fixed at indicated times and assessed by immunofluorescence. To deplete p54nrb, CARM1 or Neat1, embryos were injected at the zygote stage with a combination of three siRNAs at a total concentration of 12 μM (for p54nrb depletion) 200nM stealth siRNA (for CARM1 depletion) or 200 nM antisense oligonucleotides ASO (for Neat1 depletion). Controls were injected with 12 μM AllStars Negative Control siRNA or antisense LNA negative control and all embryos were co-injected with 200 ng μl−1Gap43-GFP mRNA as an injection control. The embryos were fixed at indicated time points and assessed by immunofluorescence or subjected to RT-qPCR. See Table S1 for sequences.
8
Microinjection of Fibrillarin mRNA in Mouse Oocytes
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Mouse fibrillarin was subcloned into the RN3P vector for in vitro transcription of mRNA. Capped mRNAs were generated using a T3 mMESSAGE mMACHINE Kit (Thermo Fisher Scientific, AM1348) following the manufacturer’s instructions. Microinjection of mRNA at desired concentrations into mouse GV oocytes was performed on a Leica DMI3000B microscope equipped with a Leica micromanipulator as previously described (Na and Zernicka-Goetz, 2006 (link)).
9
Glass Capillary Needle Fabrication
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Needles were made from glass capillaries (25-μl Drummond Microcaps) using a needle puller (Narishige), and the tip was sharpened using a needle grinder (Narishige). The needle gauge was adjusted to 12–14 μm. A micromanipulator (Leica) equipped with a glass needle was used for transplantation.
10
Cell Cycle Synchronization and Fractionation
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For cell-cycle synchronization, mESC (J1) cells124 (link) were treated with 1.25 mM Thymidine for 14 h and then 50 ng/mL Nocodazole for 7 h. G1 and S phase cells were collected at 1.5 h and 7 h, respectively, after Nocodazole release. mESCs were treated with DRB (100 μM) and ActD (1 μg/mL) for 3 h to inhibit transcription. For heterochromatin fractionation, sucrose gradient centrifugation of mESC nuclear extracts was performed as previously reported with modifications.125 (link),126 (link) For embryonic microinjection, ASO (5 μM) and AMO (1 mM) was injected into PN3 zygotes on a Leica DMI3000B microscope equipped with a Leica micromanipulator.
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