Intravitreal injections were performed according to a published protocol (Qian et al., 2008 (link)). All procedures were performed under dim-red light. Dark-adapted mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (6 mg/kg), and one drop of 0.5% tetracaine topical anesthetic was applied to the cornea. One microliter of the drug (MFA, 200 μM) was injected into the mouse vitreous body. Injections were done through the sclera on the nasal side of the eye approximately 1 mm posterior to the limbus with a 10-μl Nanofil syringe with a removable 35-gauge needle (World Precision Instruments, Inc., Sarasota, FL, USA). Intravitreal concentrations of MFA were estimated to be 10 μM by assuming a complete mixture and the vitreal volume to be ~20 μl for a mouse eye (Wang et al., 2015 (link)). Phosphate buffer saline (PBS) was used as an injection control. ERG recordings were performed 2 h after injection with animals kept in dark.
Removable 35 gauge needle
Manufactured by World Precision Instruments
Sourced in United States
The Removable 35-gauge Needle is a precision laboratory instrument designed for delicate tasks. It features a removable 35-gauge tip, allowing for versatile application and easy maintenance.
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3 protocols using removable 35 gauge needle
1
Intravitreal Injection Protocol for Mouse
2
Intravitreal Gene Therapy for Rs1-KO Mice
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Recombinant AAV-RS1 gene therapy vector described previously32 (link) was unilaterally administered into 28 Rs1-KO mice on postnatal day 21 to 26 by intravitreal injection; the contralateral eye remained untouched. Rs1-KO mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and one drop of 0.5% tetracaine topical anesthetic was applied to the cornea. One microliter of viral vector at a dose of 2.5 × 109 vector genomes per eye was injected into the vitreous body through the sclera on the nasal side of the eye approximately 1 mm posteriorly to the limbus with a 10-μL Nanofil syringe with a removable 35-gauge needle (World Precision Instruments, Inc., Sarasota, FL, USA). After injection, triple antibiotic ophthalmic ointment (neomycin, polymyxin B, and bacitracin) was applied to the eye.
3
Intravitreal AAV-RS1 Gene Therapy
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Recombinant AAV-RS1 gene therapy vector was administered into one eye of 55 Rs1-KO mice on postnatal day 21 to 26 by intravitreal injection at 2.5 × 109 vector genomes per eye, as described previously21 (link); the contralateral eye remained untouched. The viral vector was injected into the vitreous body through the sclera on the nasal side of the eye approximately 1 mm posteriorly to the limbus with a 10-μL Nanofil syringe and a removable 35-gauge needle (World Precision Instruments, Inc., Sarasota, FL, USA). After injection, triple antibiotic ophthalmic ointment (neomycin, polymyxin B, and bacitracin) was applied to the eye.
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