All mice were kept on a standard 12-h light/dark cycle and allowed food and water ad libitum. All experiments were carried out in accordance with animal care guidelines stipulated by the Animal Care and Use Committee at the University of Southampton and the Home Office (PPL 30/3095). The anaesthetic used for all mice consisted of a combination of 10 mg/ml ketamine (Pfizer) with 1 mg/ml xylazine (Bayer), injected intraperitoneally 0.1 ml per 10 g body weight. The glass capillary micropipette routinely used for our intracerebral injections was purchased from Sigma UK and the tip was adjusted to a diameter of <50 μm using a Sutter P97 Flaming Brown Pipette puller. The exact volumes and concentrations for intracerebral injections are described below. Mice were culled at 5 min after withdrawal of the glass capillary through overdose with pentobarbital 200 mg/kg. Mice were intracardially perfused with 0.1 M piperazine-N,N′-bis(2-ethanesulfonic acid) buffer (PIPES, PH 7.2) followed by 3.4 % formaldehyde plus 3 % glutaraldehyde in 0.1 M PIPES buffer. Brains were removed through dissection from the skull, post-fixed overnight in fresh fixative and then sectioned into 200 μm thick coronal slices using a vibratome.
Sutter p 97 flaming brown pipette puller
Manufactured by Sutter Instruments
Sourced in United States
The Sutter P-97 Flaming Brown pipette puller is a laboratory instrument used to create customized pipettes from glass or quartz capillary tubes. The device utilizes heat and mechanical force to pull and shape the capillary material into the desired pipette form.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using sutter p 97 flaming brown pipette puller
1
Intracerebral Injection Protocol in Mice
2
Ion-Selective Microelectrode Measurements
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SIET was used to measure K+ activities at the skin and ionocyte surfaces of larvae. Glass capillary tubes (no. TW 150-4, World Precision Instruments, Sarasota, FL) were pulled on a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA) into micropipettes with tip diameters of 3–4 μm. These were then baked at 120 °C overnight and coated with dimethyl chlorosilane (Sigma-Aldrich) for 3 h. The micropipettes were backfilled with a 1-cm column of electrolytes and frontloaded with a 50-μm column of liquid ion-exchange cocktail (Sigma-Aldrich) to create an ion-selective microelectrode (probe). The following ionophore cocktails (and electrolytes) were used: potassium ionophore I - cocktail B (100 mM KCl) and NH4+ ionophore I cocktail B (100 mM NH4Cl). To calibrate the ion-selective probe, the Nernstian property of each microelectrode was measured by placing the microelectrode in a series of standard solutions (0.1, 1, 10, and 100 mM KCl for the K+ probe; 0.1, 1, and 10 mM NH4Cl for the NH4+ probe). By plotting the voltage output of the probe against log[K+] and log[NH4+]values, a linear regression yielded a Nernstian slope of 59.1 ± 0.5 (n = 10) for K+ and 58.6 ± 0.8 (n = 10) for NH4+.
3
SIET Technique for Zebrafish H+ Flux
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In the present study, we used the SIET technique to detect H+ flux at the surface of zebrafish larvae. The method was performed largely as described previously (Shih et al., 2008 (link), 2012 (link)). In brief, micropipettes with tip diameters of 3–4 μm were pulled by a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA). Micropipettes were then baked at 120°C overnight and coated with dimethyl chlorosilane (Sigma-Aldrich) for 30 min. To make an ion-selective microelectrode (probe), micropipettes were backfilled with a 1-cm column of electrolytes and frontloaded with a 20–30-μm column of liquid ion-exchange cocktail (Sigma-Aldrich). The following ionophore cocktail and electrolytes were used: H+ ionophore I cocktail B (40 mM KH2PO4 and 15 mM K2HPO4; pH 7). To calibrate the ion-selective probe, the Nernstian property of each microelectrode was measured by placing the microelectrode in a series of standard solutions (pH 6, 7, and 8 for the H+ probe). By plotting the voltage output of the probe against [H+] values, a linear regression yielded a Nernstian slope of 58.6 ± 0.8 (n = 10) for H+.
4
Fabrication of Ca2+ Selective Microelectrodes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To construct ion-selective microelectrodes, glass capillary tubes (no. TW 150–4; World Precision Instruments, Sarasota, FL, USA) were pulled on a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA, USA) into micropipettes with tip diameters of 3~4 μm. The micropipettes were then baked at 120°C overnight and coated with dimethyl chlorosilane (Sigma-Aldrich) for 30 min. The micropipettes were backfilled with a 1-cm column of 100 mM CaCl2 for the Fluka Ca2+-selective microelectrode. The microelectrode was then frontloaded with a 20~30-μm column of Ca2+ ionophore I cocktail A (Sigma-Aldrich) to create a Ca2+-selective microelectrode.
5
Measuring Na+ Flux in Medaka Larvae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SIET was used to measure Na+ flux activity at the epithelium surface of medaka larva. Glass capillary tubes (no. TW 150 – 4; World Precision Instruments, Sarasota, FL) were pulled on a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA) into micropipettes with tip diameters of 3–4 μm. The micropipettes were then baked at 120 °C overnight and coated by incubation with dimethyl chlorosilane (Sigma-Aldrich) for 30 min. The micropipettes were backfilled with a 1-cm column of electrolytes and frontloaded with a 20–30 μm column of liquid ion-exchange cocktail (Sigma-Aldrich) to create an ion-selective microelectrode (probe). The ionophore cocktail (and electrolytes) was Na+ ionophore II cocktail A (100 mM NaCl). To calibrate the ion-selective probe, the Nernstian response of each microelectrode was evaluated by placing it in a series of standard solutions (0.1, 1, and 10 mM NaCl dissolved in distilled water). By plotting the voltage output of the probe against log [Na+] value, linear regression yielded a Nernstian slope of 56.7 ± 0.5 (N = 10). In preliminary tests, the selectivity of the Fluka Na+ ionophore II cocktail A was 10–16 times more selective to Na+ than to NH4+ (measured in 1–10 mM Na+ solution).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!