27 g needle

Hatched silkworm larvae were raised by feeding an artificial diet containing antibiotics (Silk Mate 2S) in an incubator at 27 °C until the fourth molting stage. On the first day of fifth-instar larvae, silkworms (n = 5) were fed Silk Mate 2S. On the second day, mycobacterial suspensions (6.0 × 10⁷ to 7.0 × 10⁸ CFU/larva) were injected into the hemolymph through the dorsal surface of the silkworm using a disposable 1-mL syringe with a 27-G needle (TERUMO, Tokyo, Japan). After injection, the number of silkworms that survived was counted at the indicated time until 80 h. The data are plotted according to the Kaplan–Meier method [30 (link)].

Oocytes were obtained from ovaries 44–48 hr following intra‐peritoneal injection of 5 international units (IU) of pregnant mare's serum gonadotropin (PMSG; Pacificvet) as described before (Gui & Homer, 2012, 2013; Homer, Gui, & Carroll, 2009; Wei, Greaney, Zhou, & Homer, 2018; Zhou, Hancock, Khanna, & Homer, 2019). Following humane euthanasia, ovaries were dissected and antral follicles were punctured using a 27 G needle (Terumo) under direct vision of a stereomicroscope (SMZ‐800N; Nikon) to release oocytes into pre‐warmed αMEM HEPES‐buffered medium supplemented with 50 μM 3‐isobutyl‐1‐methylxanthine (IBMX; Sigma), which prevents oocytes from undergoing GVBD. To study maturation, oocytes were allowed to resume meiosis by culturing them in micro‐drops of IBMX‐free M16 culture medium (Sigma) under embryo‐tested mineral oil (Sigma) at 37°C in an atmosphere of 5% CO₂ and 5% O₂. Generation of histone 2B (H2B)‐RFP cRNA (250 ng/µl) and microinjection into GV‐stage oocytes was performed as described previously (Subramanian et al., 2020; Wei et al., 2018; C. Zhou et al., 2019).

C2C12 myotubes were treated with DMSO (0.1% vol/vol) or TAK-242 (1 μM) and then with PBS or LPS (1 μg/mL) 1 h later. After 4 h cell culture, media were collected and centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was removed and stored at −80 °C until tested.
Mice were injected with vehicle or TAK-242 (3 mg/kg) and then with PBS or LPS (1 mg/kg) 1 h later. After 4 h, mice were anaesthetised by the i.p. injection of medetomidine (0.003%; Domitor, Nippon Zenyaku, Koriyama, Japan), midazolam (0.04%; Dormicum, Astellas Pharma, Tokyo, Japan), and butorphanol tartrate (0.05%; Vetorphale, Meiji Seika Pharma, Tokyo, Japan) dissolved in sterile 0.9% NaCl. Blood samples were withdrawn from the inferior vena cava using a 27 G needle (Terumo, Tokyo, Japan) into EDTA tubes, which were centrifuged at 3000 rpm for 15 min at 4 °C. The plasma layer was removed and stored at −80 °C until tested.
IL-6 and TNF-α concentrations in culture supernatant and plasma were determined using mouse IL-6 and TNF-α ELISA kits (Proteintech, Rosemont, IL), respectively, according to the manufacturer’s protocol. Absorbance at 450 nm was measured using a spectrophotometer (Thermo Fisher Scientific).

The ovarian tissue vitrification was carried out using a needle immersion vitrification protocol described previously [9 (link)] with slight modification. Briefly, ovarian tissues were threaded onto a 27 G needle (5–6 pieces per needle; Terumo Corporation, Tokyo, Japan) with space between each piece. The needles that pierced the tissue were rinsed in KAv-1 medium at first and immersed in equilibration solution consisting of 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG) in KAv-1 medium for 10 min at 4 °C. The needles were subsequently moved into vitrification solution consisting of 15% DMSO, 15% EG, and 0.5 M sucrose in KAv-1 for 10 min at 4 °C. Then, after excess solution was absorbed quickly with a Kimwipe, the needles were plunged into liquid nitrogen directly, inserted into a cryovial (Watson, Tokyo, Japan) containing liquid nitrogen, and stored for at least 1 week.
For warming, needles holding tissue pieces were taken out of the cryovial in liquid nitrogen and immediately transferred into a washing gradient solution. The vitrified tissues held by the needles were washed in gradient solution (KAv-1 supplemented with 1, 0.5, 0.25, and 0 M sucrose) for 5 min at each step at 37 °C. The ovarian tissues were then removed from the needles for further assessment.

Protein concentration of 1% SDS (AppliChem, Leuven, Belgium) protein extracts sheared with a 27 G needle (Terumo, Leuven, Belgium) was determined using a bicinchoninic acid (BCA) protein assay (Pierce, Aalst, Belgium). Proteins were separated on a 12.5% w/v SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (PVDF; BioRad) using an XCell SureLock electrophoresis system (Invitrogen). LEDGF-hybrids were detected using 1/2.000 polyclonal rabbit anti-LEDGF_480–530 antibody (A300-848a; Bethyl Laboratories-Imtec Diagnostics N.V., Antwerpen, Belgium) and 1/5 000 secondary antibody (polyclonal goat anti-rabbit antibody coupled with horse radish peroxidase (HRP); Dako). Chemiluminescence was measured using a ECL plus western blotting detection kit (Amersham Biosciences, Roosendaal, The Netherlands). Equal loading was verified with a primary monoclonal antibody directed to α-tubulin (mouse, 1/10 000, 1 h at room temperature; T5168, Sigma-Aldrich) and secondary antibody in blocking buffer (1/10 000, polyclonal goat-anti mouse labelled with HRP; Dako). Visualization was done by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo scientific).

To investigate the effects of peripherally or centrally administered orexin on cytokine production in mice with endotoxin shock, orexin was administered as described above. Monitoring body temperature, the blood and brain were sampled at 4 hr or 22 hr after LPS injection. For sampling the blood and brain, mice were anesthetized with isoflurane. Blood was sampled from the left ventricle using a 27 G needle (Terumo, Japan) with heparin or EDTA-2Na, and was centrifuged at 3000 rpm for 15 min. The supernatants were sucked-up as blood plasma or serum. Removed brains were frozen in liquid nitrogen and were stored at −80°C.