Rabbits were kept dark-adapted for at least 1 hour before surgery and retinal slice preparation. Following enucleation, under dim-red illumination, a central rectangular portion of retina was cut close to the visual streak, placed photoreceptor side down on filter paper (Millipore, Billerica, MA) before isolating the retina. Thick (250-μm) slices were prepared with a tissue chopper (Stoelting, Wood Dale, IL) and placed in a custom electrical recording chamber for use over the course of 3 hours. The retinal slices were continuously superfused at 3 mL/min with bicarbonate-based Ames medium and maintained at 34°C and pH 7.4 by bubbling it with 95% O2 and 5% CO2. The osmolality was adjusted to 285 mOsm/kg.
Tissue chopper
Manufactured by Stoelting
Sourced in United States
The Tissue Chopper is a laboratory instrument designed to precisely cut tissue samples into thin, uniform slices. It is a versatile tool used in various fields of study, such as biology, histology, and neuroscience, where the analysis of thin tissue sections is crucial.
Automatically generated - may contain errors
Lab products found in correlation
Millicell culture insert, by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Filter paper, by Merck Group (1 mentions)
Luciferin, by Biosynth (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
6 well plate, by SPL Life Sciences (1 mentions)
Gey s balanced salt solution gbss, by Merck Group (1 mentions)
Basal medium eagle, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Mco 175, by Sanyo (1 mentions)
Mem eagle medium, by Thermo Fisher Scientific (1 mentions)
6 well culture plate, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Merck Group (1 mentions)
Zen software, by Zeiss (1 mentions)
10 protocols using tissue chopper
1
Dark-Adapted Rabbit Retinal Slice Prep
2
Hippocampal Slice Preparation from Developing Mice
3
Organotypic Hippocampal Slice Culture Preparation
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Organotypic hippocampal slice cultures were prepared as previously described [38 (link)]. Briefly, whole brain of newborn rats (postnatal days 5–7) was dissected and placed in ice-cold Ca2+-free dissection solution (10 mM D-glucose, 4 mM KCl, 26 mM NaHCO3, 8% sucrose, 5 mM MgCl2, 1:1000 Phenol Red) saturated with 5% CO2/95% O2, enabling the brain to chill for 1 min. Hippocampi were then isolated with the help of a magnifying glass, cut in slices (400 µm) in the same solution using a tissue chopper (Stoelting), and maintained at 35.5 °C in culture on permeable membranes in a medium containing 20% horse serum, 1 mM CaCl2, 2 mM MgSO4, 1 mg/L insulin, 0.0012% ascorbic acid, 30 mM HEPES, 13 mM d -glucose, and 5.2 mM NaHCO3. Culture medium was replaced with fresh one every 2–3 days. The slices were used at 4–8 days in vitro.
4
Culturing SCN Brain Slices
5
Organotypic Hippocampal Slice Culture
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Using the Stoppini method15) (link), organotypic slice cultures of the hippocampus were prepared from a total of 12 Sprague Dawley rats (DooYeol Biotech, Seoul, Korea) aged 7days. Rats were stabilized for 24 hours before the start of the experiment and were decapitated using scissors. Heads were sterilized with 70% alcohol, and the hippocampi were quickly removed and placed in ice-cold Gey's Balanced Salt Solution (GBSS, Sigma Aldrich, St Louis, MO, USA). The hippocampi were sliced (450 µm thick) using a tissue chopper (Stoelting Co., Wood Dale, IL, USA), and Millicell culture inserts (Millipore, Ireland, 6 slices per insert) were placed into 6-well plates (SPL Life Sciences Co., Pocheon, Korea) with 1 mL of Gahwiler's media per well. The slice culture media consisted of 25% Hank's Balanced Salt Solution (GibcoBRL/Life Technologies, Grand Island, NY, USA), 25% heat inactivated horse serum (Hyclone, Logan, UT, USA), 50% basal medium eagle (GibcoBRL/Life Technologies), 6.5-mg/mL glucose, and 200mM glutamax-I (GibcoBRL/Life Technologies). Slices were cultured at 37℃ with 5% CO2 in a humidified incubator (MCO175, Sanyo, Tokyo, Japan), and the media was changed twice per week. All procedures were performed aseptically in a horizontal flow hood.
6
Organotypic Rat Hippocampal Slice Culture
7
Hippocampal Slice Culture BDNF Treatment
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Hippocampal slice cultures were prepared as described previously (Fuller and Dailey, 2007 (link)) from P5 rat pups. The hippocampus was removed from the brain and 400 µm thick slices were cut transversely from both hippocampi in ice-cold Hank’s balanced salt solution (HBSS) containing glucose, using a tissue chopper (Stoelting). BDNF (100 ng/ml) was added to the slice culture media at DIV14 for 5 min or 48 h incubation. After treatment with 100 ng/ml BDNF for 5 min or 48 h, slices were washed once and homogenized in 320 mM sucrose, 4 mM HEPES-KOH, pH 7.4, in a glass-Teflon homogenizer with 10 strokes at 900 rpm. Protein concentration was determined using a BCA kit (Calbiochem, cat. no. 71285-3) and equal protein amounts were subjected to SDS-PAGE followed by immunoblotting. Antibodies used for Western blotting were mouse monoclonal anti-Rab3a and anti-tubulin, and mouse monoclonal anti-VAMP2 (all from Synaptic Systems).
8
Organotypic Hippocampal Slice Culture
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Organotypic hippocampal slices were prepared from 5-7-day-old C57Bl6 pups according to Stoppini et al. 1991 [26 (link)] with slight modifications as described in [13 (link)]. Briefly, the hippocampi were extracted in carbonated low Na cerebrospinal fluid (CSF) (1 mM CaCl2, 10 mM D-glucose, 4 mM KCl, 5 mM MgCl2, 26 mM NaHCO3, 234 mM sucrose and 0.1% phenol red solution) and coronal slices of 400 μm were made using a tissue chopper (Stoelting, #51425). Hippocampal slices with intact dentate gyrus (DG) and cornu ammonis (CA) regions were selected and maintained on air-fluid interface-style Millicell culture inserts, 30 mm diameter, 0.4 μm (Millipore, #PICM0RG50) in 6-well culture plates (ThermoFisher Scientific) with 800 μL of 37°C pre-heated sterile medium (MEM Eagle medium 78.8% (Gibco #11095), 20% heat-inactivated horse serum (Gibco, #16050–122), 1 mM L-glutamine, 1 mM CaCl2, 2 mM MgSO4, 170 nM insulin, 0.0012% ascorbic acid, 12.9 mM D-glucose, 5.2 mM NaHCO3, 300 mM Hepes (Sigma #H3375), pH = 7.28, osmolality adjusted to 317–322). The medium was replaced completely three times per week.
9
Organotypic Hippocampal Slice Culture Preparation
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Organotypic hippocampal slice cultures were prepared as previously described [38 (link)]. Briefly, whole brain of newborn rats (postnatal days 5–7) was dissected and placed in ice-cold Ca2+-free dissection solution (10 mM D-glucose, 4 mM KCl, 26 mM NaHCO3, 8% sucrose, 5 mM MgCl2, 1:1000 Phenol Red) saturated with 5% CO2/95% O2, enabling the brain to chill for 1 min. Hippocampi were then isolated with the help of a magnifying glass, cut in slices (400 µm) in the same solution using a tissue chopper (Stoelting), and maintained at 35.5 °C in culture on permeable membranes in a medium containing 20% horse serum, 1 mM CaCl2, 2 mM MgSO4, 1 mg/L insulin, 0.0012% ascorbic acid, 30 mM HEPES, 13 mM d -glucose, and 5.2 mM NaHCO3. Culture medium was replaced with fresh one every 2–3 days. The slices were used at 4–8 days in vitro.
10
Immunohistochemistry of Mouse Hippocampal Slices
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Acute hippocampal slices were prepared from 8-week-old mice anesthetized with isoflurane and decapitated. The hippocampus was removed and 200 mm thick slices were cut transversely using a tissue chopper (Stoelting) in ice-cold artificial cerebrospinal fluid (ACSF) containing, in mM 124 NaCl, 4.9 KCl, 1.2 KH 2 PO 4 , 2 MgSO 4 , 2 CaCl 2 , 24.6 NaHCO 3 , and 10 D-glucose (saturated with 95% O 2 and 5% CO 2 , pH 7.4, ~305 mOsm). Slices were fixed in 4% paraformaldehyde in PBS for 30 min and washed 3 X 20 min in PBS. After washing, slices were incubated in antibody buffer (2% donkey serum, 0.1% Triton X-100 and 0.05% NaN 3 in PBS) for 30 min at room temperature. Then slices were incubated with primary antibodies in antibody buffer overnight at 4°C. Slices were then washed with PBS 3 X 20 min and incubated with fluorescently tagged secondary antibodies for 2 hours at room temperature. Slices were washed 3 X 20 min in PBS and mounted on microscope slides with Fluoromount-G (Sigma) and sealed with nail polish. Images were collected using 10X air and 40X oil immersion objectives on a Zeiss A1 laser scanning confocal microscope with Zen software (Carl Zeiss). Digital images were processed using Adobe Photoshop software.
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