HH10 embryos were incubated with 0.25% Trypsin-EDTA at 38°C for 7 min. Trypsin was then de-activated by transferring into 20% chick serum on ice for 5 min. Embryos were then washed with Tyrode's solution and pinned onto Sylgard-coated dissection dishes. Head surface ectoderm and peri-ocular mesenchyme were carefully removed using 30 gauge syringe needles from both dorsal and ventral sides. Once cleaned, both optic vesicles were removed and held in Tyrode's solution on ice. Left and right optic vesicles were separately pooled from at least five embryos, yielding two match-paired pools for use as treated and control samples. Pooled vesicles were cultured in polyHEMA (Sigma)-coated culture wells to prevent adhesion, with DMEM-F12 media (Invitrogen) supplemented with 1× N2 (Invitrogen), 1× L-glutamate and 1× penicillin/streptomycin at 37°C and 5% CO2 for 16 h. Culture media for treated samples was supplemented with the following factors as required: 35 ng/ml Bmp4 (R&D Systems), 0.5 µM BIO (Sigma) with 0.1% DMSO (Sigma), 10 µM SIS3 with 0.1% DMSO (Sigma), or 2.5 µM cyclopamine (Sigma) with 0.1% 2-hydroxypropyl-β-cyclodextrin (HBC; Sigma).
2 hydroxypropyl β cyclodextrin hbc
Manufactured by Merck Group
Sourced in United States
2-hydroxypropyl-β-cyclodextrin (HBC) is a modified cyclodextrin compound. Cyclodextrins are cyclic oligosaccharides composed of glucose units. HBC is used as a pharmaceutical excipient to enhance the solubility, stability, and bioavailability of various drug compounds.
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Lab products found in correlation
Cyclodextrin encapsulated e2, by Merck Group (2 mentions)
D12492, by Research Diets (2 mentions)
Cyclopamine, by Merck Group (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Poly hema, by Merck Group (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Clozapine n oxide cno, by Cayman Chemical (1 mentions)
Kds 100, by KD Scientific (1 mentions)
Gw4064, by Merck Group (1 mentions)
Letrozole, by Selleck Chemicals (1 mentions)
9 protocols using 2 hydroxypropyl β cyclodextrin hbc
1
Optic Vesicle Isolation and Culture
2
Estradiol Infusion and DREADD Activation in Memory Consolidation
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Infusions into the mPFC or DH were conducted as described previously (Kim et al., 2016 (link); Tuscher et al., 2016b (link)). Briefly, cyclodextrin-encapsulated 17β-E2 (Sigma-Aldrich) was dissolved in sterile 0.9% saline to a concentration of 10 µg/µl, and infused bilaterally into the DH or mPFC immediately after training. The vehicle, 2-hydroxypropyl-β-cyclodextrin (HBC; Sigma-Aldrich), was dissolved in saline to the same concentration of cyclodextrin present in the cyclodextrin-encapsulated E2 solution. Infusions were conducted at a rate of 0.5 μl/min for 1 min per hemisphere as described previously (Fernandez et al., 2008 (link); Fortress et al., 2013 (link)), resulting in a dose of 5-µg E2/hemisphere.
For DREADD experiments, stock solutions of clozapine-N-oxide (CNO, Cayman Chemical) were prepared by dissolving CNO in 100% dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml, and storing 10-µl aliquots at –20°C, as described previously (Tuscher et al., 2018 (link)). On the day of injection, CNO stock was thawed and diluted to 1 mg/ml in a solution of sterile 0.9% saline containing 2% DMSO. Intraperitoneal injections of 1-mg/kg CNO were administered immediately after training, followed directly by bilateral DH infusion of vehicle or E2.
For DREADD experiments, stock solutions of clozapine-N-oxide (CNO, Cayman Chemical) were prepared by dissolving CNO in 100% dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml, and storing 10-µl aliquots at –20°C, as described previously (Tuscher et al., 2018 (link)). On the day of injection, CNO stock was thawed and diluted to 1 mg/ml in a solution of sterile 0.9% saline containing 2% DMSO. Intraperitoneal injections of 1-mg/kg CNO were administered immediately after training, followed directly by bilateral DH infusion of vehicle or E2.
3
Cyclopamine Administration in Pregnant Mice
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Pregnancies were timed by the detection of vaginal plugs, corresponding to E0.5. Pregnant mice were given daily i.p. injections of 2 mg/kg cyclopamine (Stemgent) complexed with 2-hydroxypropyl-β-cyclodextrin (HBC; Sigma) from day E15.5 to day 20 (van den Brink et al., 2001 (link)). A cyclopamine-HBC stock solution was produced by suspending 1 mg cyclopamine in 1 mL 45% HBC in sterile PBS and stirring for 60 min at 65°C. The cyclopamine-HBC stock was stored at −20°C until use. Mice were injected i.p. daily with 10 mg/kg of body weight, for six days. Six independent experiments were run, each comprising three to five Pdx1-Cre;αE-catenin-KO and five to eight WT animals.
4
Hippocampal Estrogen Infusion Protocol
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All mice were infused with vehicle or E2. Cyclodextrin-encapsulated E2 (Sigma-Aldrich) was dissolved to a concentration of 5 μg/0.5 μL in physiological saline as described previously (Fortress et al. 2013b (link)). The vehicle, 2-hydroxypropyl-β-cyclodextrin (HBC, Sigma-Aldrich), was dissolved in saline to the same concentration of cyclodextrin used in the cyclodextrin–E2 solution. During infusions, mice were gently restrained and dummy cannulae were replaced with infusion cannulae (C232I, 26 gauge extending 0.8 mm beyond the 1.5-mm guide cannulae), which were attached to PE50 tubing connected to a 5-μL Hamilton syringe. Using a microinfusion pump (KDS 100, KD Scientific), mice were bilaterally infused into the dorsal hippocampus at a rate of 0.5 μL/min for 1 min, resulting in an E2 dose of 5 μg/hemisphere. Infusion cannulae remained in place for 1 min to prevent diffusion back up the cannula track. Infusions occurred immediately after behavioral training (i.e., post-training). Infusions were spaced out by 2 wk to allow the acute effects of E2 to dissipate, and all mice received the same number of infusions.
5
Dietary Regulation of FXR Deficiency
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10 weeks age KK-Ay mice and C57BL/6J, homozygous FXR deficient (FXR–/–) mice were maintained under environmentally controlled conditions with free access to standard chow diet and water as described previously28 (link). Animal experiments were conducted in the barrier facility of the Laboratory Animal Center, Xiamen University, approved by the Institutional Animal Use and Care Committee of Xiamen University, China, and the methods were carried out in accordance with the approved guidelines. Mice were fed with high fat diet containing 60% fat, 20% protein and 20% carbohydrate (Research Diets, D12492, New Brunswick, USA), and the food intake were measured every second day. Mice were i.p. injected with either vehicle (40% of 2-hydroxypropyl-β-cyclodextrin (HBC), Sigma, USA) or vehicle containing GW4064 (30 mg/kg, Sigma) or avermectin analogues (1.3 mg/kg) (International Laboratory, USA) once a day for 14 days. After 6 h fasting, mice were weighed and sacrificed. Livers were weighed and taken photos, and part of each liver was fixed in 4% paraformaldehyde. Liver histology characterization was analyzed by haematoxylin and eosin (H&E) staining with paraffin-embedded sections by standard procedures. Other tissues were collected and frozen in liquid nitrogen for use, and serums were collected to measure metabolite parameters.
6
Vidofludimus Improves Obesity in ob/ob Mice
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10-11 weeks old male obese Lepob/ob C57BL/6 (ob/ob) mice were purchased from the Model Animal Research Center of Nanjing University, China. Mice were randomly assigned to experimental groups. Mice were intraperitoneally (i.p.) injected with either vehicle (40% of 2-hydroxypropyl-β-cyclodextrin (HBC), Sigma, USA) or vehicle containing vidofludimus (10 mg/kg) once daily for 14 days(n = 6 per group). After fasting for 6 h, mice were weighed and sacrificed by cervical dislocation. Liver histology characterization was analyzed by H&E staining with paraffin-embedded sections. Fresh liver tissues were embedded in optimum cutting temperature compound (OCT) and cryo-sectioned. The sections were fixed in 4% paraformaldehyde in PBS, and stained with 0.3% oil red O. Other tissues were collected and frozen in liquid nitrogen for use, and plasma were collected to measure metabolite parameters.
7
Metabolic Regulation in Diabetic Mice
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Animal experiments were performed according to procedures approved by the Institutional Animal Use and Care Committee of Xiamen University. 8–10 week-old male mice (db/+, db/db and KKAy mice from Hua Fukang, Beijing, China) were acclimatized for 7 days under standard conditions before experiments. Mice were fed a high-fat diet (60% kcal fat, D12492, Research Diets Inc, USA) and intraperitoneally (i.p.) injected once daily with vehicle (40% of 2-hydroxypropyl-β-cyclodextrin, HBC) (Sigma, USA), 3 mg/kg of chelerythrine or 3 mg/kg of rosiglitazone for 14 days. Mice were euthanized after 6 h of fasting, and serum samples were collected to measure the metabolic parameters.
8
Letrozole Infusion into Dorsal Hippocampus
9
Cyclopamine Administration in Pregnant Mice
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