Adults were aged at 20 °C for 5–7 days before performing antennal ablation (Paglione et al., 2020 (link)). Unilateral antennal ablation (e.g., removal of one antenna) was performed using high precision and ultra-fine tweezers, and flies returned to vial for the appropriate time. The ablation of 3rd antennal segments did not damage the rest of the head or lead to fly mortality. At corresponding time points, adult brain dissections were performed as described (Paglione et al., 2020 (link)): decapitated heads were fixed in 4% formaldehyde in PTX (0.5% Triton X-100 in PBS) for 20 min, and washed 3x10 min with PTX. Brain dissections were performed in PTX, and dissected brains were fixed in 4% formaldehyde in PTX for 10 min, followed by 1 hr of blocking in 10% normal goat serum (Jackson Immuno) in PTX and an O/N incubation with the following primary antibodies at 4 °C in blocking solution: 1:500 chicken anti-GFP (Rockland), and 1:150 mouse anti-nc82 (DSHB, nc82). Brains were then washed 3x10 min with PTX at RT, and incubated with secondary antibodies in PTX at RT for 2 hr: 1:200 Dylight 488 goat anti-chicken (abcam, ab96947), and 1:200 AlexaFluor 546 goat anti-mouse (ThermoFisher, a-11030). Brains were washed 3x10 min with PTX at RT, and mounted in Vectashield for microscopy.
Ab96947
Manufactured by Abcam
AB96947 is a lab equipment product from Abcam. It is a device designed for scientific research applications. The core function of this product is to provide a controlled environment for scientific experiments or sample preparation.
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4 protocols using ab96947
1
Unilateral Antennal Ablation and Fly Brain Dissection
2
Immunohistochemical Labeling of Brain Sections
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Animals were deeply anaesthetized with 15 mg pentobarbital (i.p.) and the heart transfused with 20 ml ice cold heparinized PBS followed by 30 ml 4% formalin. Brains were removed and post-fixed overnight in 4% formalin. They were then cryoprotected in 40% sucrose for 24–48 hr. Sections of 30 µm were cut in a cryostat. Free-floating sections were washed in PBS plus 0.1% Triton X-100 (PBS-T) three times for 10 min each and then blocked by incubation with 4% bovine serum albumin in PBS-T for 1 h. Free floating sections were incubated with primary antibodies for GFP (Life Technologies: A10262; 1:4000) for 24–48 h at 4 °C. They were then washed in PBS-T, three times for 10 min each and then incubated with secondary antibody (Abcam: AB96947, 1:500) for 1 h at room temperature. Confirmation of electrode placement was performed in brain sections stained with bisBenzimide as a counter stain to the DIO that coated the electrodes. Briefly, free-floating brain slices were exposed to bisBenzimide (1 µg/ml) in PBS for 15 min at room temperature. Three washes of 15 min each in PBS were then performed and slices then mounted on glass slides and allowed to dry. A cover slip was placed on the slices with a mounting medium and then imaged on a confocal fluorescent microscope.
3
Immunohistochemical Characterization of Neuronal Populations
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Animals were deeply anaesthetized with 15 mg pentobarbital (i.p.) and the heart transfused with 20 ml ice cold heparinized PBS followed by 30 ml 4 % formalin. Brains were removed and post-fixed overnight in 4 % formalin. They were then cryoprotected in 40 % sucrose for 24 - 48 hours. Sections of 30 µm were cut in a cryostat. Free-floating sections were washed in PBS plus 0.1 % Triton X-100 (PBS-T) three times for 10 minutes each and then blocked by incubation with 4% bovine serum albumin in PBS-T for 1 hour. Free floating sections were incubated with primary antibodies for GFP (Life Technologies: A10262; 1:4000) for 24 - 48 hours at 4 °C. They were then washed in PBS-T, three times for 10 minutes each and then incubated with secondary antibody (Abcam: AB96947, 1:500) for 1 hour at room temperature.
Confirmation of electrode placement was performed in brain sections stained with bis-Benzimide as a counter stain to the DIO that coated the electrodes. Briefly, free-floating brain slices were exposed to bis-Benzimide (1 ug/ml) in PBS for 15 minutes at room temperature. Three washes of 15 minutes each in PBS were then performed and slices then mounted on glass slides and allowed to dry. A cover slip was placed of the slices with a mounting medium and then imaged on a confocal fluorescent microscope.
Confirmation of electrode placement was performed in brain sections stained with bis-Benzimide as a counter stain to the DIO that coated the electrodes. Briefly, free-floating brain slices were exposed to bis-Benzimide (1 ug/ml) in PBS for 15 minutes at room temperature. Three washes of 15 minutes each in PBS were then performed and slices then mounted on glass slides and allowed to dry. A cover slip was placed of the slices with a mounting medium and then imaged on a confocal fluorescent microscope.
4
Immunohistochemical Characterization of Neuronal Populations
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