Mice were maintained in darkness overnight before being sacrificed. After the cornea and lens were removed, a piece of retina was flattened on a membrane filter (Merck Millipore, HAWP01300). Then the whole-mounted retina was incubated in Ames’ medium and maintained at 31°C to 33°C.33 (link) TdTomato-expressing cells were identified with a brief snapshot of fluorescence excitation light.34 (link) An intracellular electrophysiological recording was then performed at tdTomato-positive cells. Light spots at different size gradients with a 2-second ON and 8-second OFF repeated three times and generated by a customized projector were casted over the retina through the 40× objective lens. The responses to spots were recorded under the current clamp. The intracellular solution contained: 121 mM potassium gluconate, 4 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 4 mM MgATP, 0.3 mM Na3GTP, 5 mM sodium phosphocreatine, and 13.4 mM biocytin (pH adjusted to 7.25 with KOH). The light intensity was calibrated to 6.2 log photons/µm2/s.24 (link),26 (link) The latency time to peak response is defined by the time from light onset or offset to peak response.
Hawp01300
Manufactured by Merck Group
Sourced in United States, United Kingdom
The HAWP01300 is a lab equipment product from Merck Group. It is designed for laboratory use, but no further details about its core function are available at this time.
Automatically generated - may contain errors
Lab products found in correlation
A0701, by Merck Group (1 mentions)
A1420, by Merck Group (1 mentions)
Vt1200, by Leica (1 mentions)
Ketamine, by Bayer (1 mentions)
Xylazine hydrochloride, by Bayer (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Oxybuprocaine hydrochloride, by Alcon (1 mentions)
Tetracaine hydrochloride, by Alcon (1 mentions)
7 protocols using hawp01300
1
Whole-Mount Retina Electrophysiology
2
Retinal Slice Preparation from tdTomato Mice
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DAT-tdTomato mice that were identified by genotyping were sacrificed and eyeballs were removed. The cornea and lens were removed to isolate the retinas. Retinal slices were made according to the previous research.27 (link) Briefly, a part of the isolated retina was embedded in 2.5% agarose (Sigma-Aldrich, A0701, St. Louis, MO), which was dissolved in the buffer for agarose gel (NaCl 119 mM, HEPES 40 mM, NaH2PO4 1.25 mM, KCl 2.5 mM, CaCl2·2O 1.15 mM and MgSO4 1.5 mM) at 150°C and held at 37°C. This embedded retina was quickly cooled and stuck at the base of the slicer (Leica Biosystems, VT1200s, Wetzlar, Germany). We cut 200-µm-thick slices out and kept them in Ames' medium (Sigma-Aldrich, A1420) saturated with carbogen (95% O2 and 5% CO2). Retinal slices were stored in oxygenated Ames' medium at room temperature (RT). For whole-mounted retinas, a piece of retina was cut out and mounted with the ganglion cell side facing up onto a filter paper (Merck Millipore, HAWP01300, Burlington, MA) with a hole in the center.
3
Retinal Dissection and Mounting
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Mice were dark-adapted for a minimum of 30 min. After cervical dislocation, eyes were gently touched with a soldering iron (Weller, BP650) to label the nasal part of the cornea and then enucleated. Retina isolation was done under deep red illumination in Ringer’s medium (110 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1.6 mM MgCl2, 10 mM d -glucose, and 22 mM NaHCO3, bubbled with 5% CO2/95% O2, pH 7.4). The retinas were then mounted ganglion cell side up on filter paper (Millipore, HAWP01300) that had a 3.5-mm-wide rectangular aperture in the center, and superfused with Ringer’s medium at 32° to 36°C in the microscope chamber for the duration of the experiment.
4
Angiogenesis Promotion by Tβ4 in hASCs
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All procedures were approved by the Institutional Animal Care and Use Committee of Korea University College of Medicine for animal research (27 Jun 2016; No. KOREA-2016-0124). Male C57BL/6J mice were obtained from Orient Experimental Animal Laboratory (Gyeonggi, Korea).
Male C57BL/6J mice randomly assigned to the following three groups (n = 3 mice per group). Window chambers (PR0001401) were covered by membrane filters (pore size = 0.45 µM; HAWP01300, both from Millipore, Burlington, MA, USA) and filled with hASCs (5 × 104) without or with 100 ng/mL of Tβ4 in 5% DMEM. All mice were anesthetized with a mixture of ketamine (44 mg/kg; Yuhan, Seoul, Korea) and xylazine hydrochloride (0.75 mg/kg; Rompun, Bayer, Leverkusen, Germany) and shaved. After skin incision, chambers were subcutaneously implanted into the dorsal side of mice. After 5 days of implantation, mice were anesthetized and images of dorsal sides of mice were taken. The number of subcutaneous microvessels per mouse was quantified.
Male C57BL/6J mice randomly assigned to the following three groups (n = 3 mice per group). Window chambers (PR0001401) were covered by membrane filters (pore size = 0.45 µM; HAWP01300, both from Millipore, Burlington, MA, USA) and filled with hASCs (5 × 104) without or with 100 ng/mL of Tβ4 in 5% DMEM. All mice were anesthetized with a mixture of ketamine (44 mg/kg; Yuhan, Seoul, Korea) and xylazine hydrochloride (0.75 mg/kg; Rompun, Bayer, Leverkusen, Germany) and shaved. After skin incision, chambers were subcutaneously implanted into the dorsal side of mice. After 5 days of implantation, mice were anesthetized and images of dorsal sides of mice were taken. The number of subcutaneous microvessels per mouse was quantified.
5
In Vitro Recording of Retinal Ganglion Cells
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For in vitro recordings of retinal ganglion cells, we used mice that had been injected with herpes-simplex virus into the Pbg or pulvinar and rabies virus into the superior colliculus to label circuit specific retinal ganglion cells as described above. For pulvinar experiments, we analyzed 64 cells from 20 Ntsr-Cre mice. For Pbg-specific ganglion cells, we recorded 50 cells in retinas from PV-Cre (N = 14) or Gad2-Cre (N = 3) mice. Retinas were isolated from mice that were dark-adapted for a minimum of 30 min. Retina isolation was done under deep red illumination in Ringer’s medium (110 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1.6 mM MgCl2, 10 mM D-glucose, 22 mM NaHCO3, bubbled with 5% CO2/95% O2, pH 7.4). The retinas were then mounted ganglion cell-side up on filter paper (Millipore, HAWP01300) that had a 3.5 mm wide rectangular aperture in the center, and superfused with Ringer’s medium at 32–36°C in the microscope chamber for the duration of the experiment.
6
PDGF-Mediated Retinal Explant Culture
7
Quantitative Impression Cytology Analysis
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The impression cytology specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine hydrochloride and tetracaine hydrochloride (Alcon Cusi S.A., Barcelona, Spain). Strips of cellulose acetate filter paper (HAWP 01300; Millipore, Bedford, MA) that were soaked in distilled water for a few hours and dried at room temperature were applied on the nasal bulbar conjunctiva adjacent to the corneal limbus, pressed gently by a forceps, and then removed. The specimens were then fixed with 10% formaldehyde, stained with periodic acid–Schiff, dehydrated in ascending grades of ethanol and then with xylene, and finally cover-slipped. Quantitative studies of conjunctival epithelial cells were conducted by taking photographs with a calibrated grid under a light microscope at a magnification of 200 × . We photographed 10 different areas of each sample selected at random. We calculated the mean individual epithelial cell area (MIECA) and nucleocytoplasmic (N/C) ratio using ImageJ software (National Institutes of Health, Bethesda, MD) and averaged the outcomes.
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