In order to induce the Cre recombinase in Cx3cr1CreER/+Rosa26DTA/+ mice, treatment with tamoxifen (TAM; Sigma, T5648-1G, St Louis, USA) was conducted when mice were 5–7 weeks old. tamoxifen was resuspended in corn oil (Sigma, C8267-500ML, St Louis, MO, USA) at 75 °C for at least 60 min. The Cx3cr1CreER/+Rosa26DTA/+ and Cx3cr1CreER/+ mice were administered 5 mg (200 μL) TAM subcutaneously on three consecutive days for microglial depletion, and then kept for one month to permit microglial repopulation, as we previously described [21 (link),22 (link)].
C8267 500ml
Manufactured by Merck Group
Sourced in United States
C8267-500ML is a laboratory product manufactured by Merck Group. It is a 500 milliliter container of the chemical substance.
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5 protocols using c8267 500ml
1
Tamoxifen-induced Microglial Depletion
2
4-OHT Intraperitoneal Injection in Mice
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A solution of 4-OHT was prepared using pure ethanol (Sigma-Aldrich, catalog no. E7023-1L) and corn oil (Sigma-Aldrich, catalog no. C8267-500ML). First, we added the 4-OHT (125 mg/ml; Sigma-Aldrich, catalog no. H6278-50 mg) to ethanol (100%; pure) and incubated this solution at 55°C with vortexing for 15 min. Then, corn oil was added to a final concentration of 4-OHT (12.5 mg/ml). Adult experimental mice were injected intraperitoneally with either 4-OHT (75 mg/kg) or vehicle once a day for four consecutive days.
3
Inducible Genetic Targeting in Mice
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As indicated throughout the study, mice of both sexes and ages (4–17 weeks) were used. Founder animals were purchased from The Jackson Laboratory for the following strains: C57BL6/J (000664), Pde6brd10/rd10 (004297), Cx3cr1CreERT2 (020940), and Cx3cr1GFP (005582).8 (link) All mice were backcrossed to the C57BL6/J background for at least 10 generations. Animals were housed and maintained in the IST Austria Preclinical Facility, with 12 h light-dark cycle and food and water provided ad libitum. All animal procedures were approved by the Bundesministerium für Wissenschaft, Forschung und Wirtschaft (bmwfw) Tierversuchsgesetz 2012, BGBI. I Nr. 114/2012 (TVG 2012) under the number GZ BMWFW-66.018/005-WF/V3b/2016 and by IST Austria Ethics Officer. For tamoxifen administration, Cx3cr1CreERT2/+ and C57BL6/J mice received intraperitoneal (i.p.) injections of tamoxifen (Sigma Aldrich, T5648-5G) dissolved in corn oil (Sigma Aldrich, C8267-500ML, 150 mg/kg body weight, 20 mg/mL stock solution) at the age of 4–6 weeks once per day for 3 consecutive days.
4
Tamoxifen-Induced Experimental CCM Lesions
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Tamoxifen (TMX) (T5648-1G, Sigma-Aldrich) was dissolved in 100% ethanol and stored at -80°C in aliquots (TMX concentration: 50 mg/ml). On the day of use, aliquoted TMX was diluted in corn oil (C8267-500ML, Sigma-Aldrich) (4 mg/ml). Neonatal pups at postnatal day 1 (P1) were injected intragastrically with 50μl of TMX (4 mg/ml) to induce experimental CCM lesions using a 30-gauge needle. A single injection of TMX at P1 was sufficient to induce CCM lesions.
5
Behavioral Assays of Tamoxifen-Induced Mice
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All behavioral tests were performed blind to mice genotype. Groups of mice were age-matched (8–16 weeks) and, prior to behavioral assays, were handled for 5 min per day for three days to reduce the stress introduced by experimenter contact. All mice were given tamoxifen (Sigma-Aldrich, #T5648) (intraperitoneal injection, 0.05 mg/g, drug concentration: 0.005 g/mL in corn oil) 6 h before behavioral testing unless indicated otherwise. In the oil control group, mice were given the same volume of corn oil (Sigma, C8267-500ML) with no tamoxifen. Behavioral tests using clozapine N-oxide (CNO; MedChemExpress, #HY-17366) were conducted in a 60-min window that began 30 min after CNO administration (intraperitoneal injection, 1 mg/kg). All behavioral tests were recorded using a video camera directly above and videos were analyzed using ANY-maze (Stoelting, USA). The ambient light was approximately 200 lux during all behavior tests.
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