All procedures with animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of the College of Pharmacy, King Saud University. Adult male Sprague-Dawley rats, 8–9 weeks of age weighing in the range of 220–250 g were overnight fasted, and streptozotocin (STZ; 65 mg/kg in 50 mM sodium citrate buffer, pH 4.5; Sigma, St. Louis, MO) was injected intraperitoneally as a single bolus. Equal volumes of citrate buffer were injected in non-diabetic control animals. Measurement of blood glucose concentrations and bodyweights were started 3 days after injection of STZ. Diabetes was confirmed by assaying the glucose concentration in blood taken from the tail vein. Rats with glucose levels >250 mg/dl were categorized as diabetic. After 4 weeks of diabetes, animals were anesthetized by intraperitoneal injection of an overdose of chloral hydrate and euthanized by decapitation. Retinas were dissected, flash frozen, and stored at −80 °C until use. Similarly, retinas were obtained from age-matched nondiabetic control rats.
Sodium citrate buffer
Manufactured by Merck Group
Sourced in United States, Germany
Sodium citrate buffer is a commonly used buffer solution in laboratory settings. It is composed of sodium citrate and citric acid, which provide a buffering system to maintain a specific pH range. The core function of sodium citrate buffer is to maintain a stable pH environment for various biochemical and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Streptozotocin (stz), by Merck Group (11 mentions)
Tween 20, by Merck Group (3 mentions)
Polysulfoethyl column, by Nest Group (3 mentions)
Phosphate buffer, by Merck Group (2 mentions)
Concentrated ammonium hydroxide, by Merck Group (2 mentions)
Enzyme kits, by Randox (2 mentions)
Normal donkey serum (nds), by Abcam (2 mentions)
Alexafluor 488 labeled donkey anti rabbit igg labeled, by Abcam (2 mentions)
Tween 20, by Abcam (2 mentions)
Glycyrrhizic acid, by Santa Cruz Biotechnology (2 mentions)
Ethanol, by Merck Group (2 mentions)
Higg1, by BioXCell (1 mentions)
Itlc sg, by Agilent Technologies (1 mentions)
17β estradiol, by Merck Group (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Ab23722, by Abcam (1 mentions)
Ab10241, by Abcam (1 mentions)
Ab8448, by Abcam (1 mentions)
Ab27684, by Abcam (1 mentions)
Dako wash buffer, by Agilent Technologies (1 mentions)
39 protocols using sodium citrate buffer
1
Streptozotocin-Induced Diabetic Rat Retina Model
2
Streptozotocin-Induced Diabetes Model
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Streptozotocin (Sigma-Aldrich, St-Louis, MO, USA) was dissolved in sodium citrate buffer (Sigma-Aldrich, pH 4.5) at a concentration of 22.5 mg/ml. A dose of 35 mg/kg was immediately injected intraperitoneally. The STZ solution must be extemporaneously prepared38 (link).
3
Radiolabeling of Anti-CAIX Antibodies
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Human IgG1 antimouse
CAIX (mCAIX, clone MSC3, Creative Biolabs) (molecular weight: 150
kDa), which reacts with both human and murine CAIX, and human IgG1-irrelevant
control antibody (hIgG1, BioXCell) (molecular weight: 150 kDa) were
conjugated in a molar ratio of 1:15 with isothiocyanatobenzyl–diethylenetriaminepentaacetic
acid (ITC–DTPA, Macrocyclis) in NaHCO3 (pH 9.5)
for 1 h at room temperature (resulting in a DTPA/antibody ratio of
3.4:1). The conjugated antibody was dialyzed against NH4Ac (0.25 M, pH 5.5) to remove the unbound chelator. DTPA-conjugated
antibodies were radiolabeled with indium-111 (111In, Curium)
after adding a twofold volume of 2-(N-morpholino)ethanesulfonic
acid (0.5 M MES, pH 5.5) buffer for 30 min at RT. Radiolabeling efficiency
exceeded 95% in all experiments, as determined by instant thin-layer
chromatography on silica gel chromatography strips (ITLC-SG, Agilent
Technologies) in 0.1 M sodium citrate buffer (Sigma Aldrich).
CAIX (mCAIX, clone MSC3, Creative Biolabs) (molecular weight: 150
kDa), which reacts with both human and murine CAIX, and human IgG1-irrelevant
control antibody (hIgG1, BioXCell) (molecular weight: 150 kDa) were
conjugated in a molar ratio of 1:15 with isothiocyanatobenzyl–diethylenetriaminepentaacetic
acid (ITC–DTPA, Macrocyclis) in NaHCO3 (pH 9.5)
for 1 h at room temperature (resulting in a DTPA/antibody ratio of
3.4:1). The conjugated antibody was dialyzed against NH4Ac (0.25 M, pH 5.5) to remove the unbound chelator. DTPA-conjugated
antibodies were radiolabeled with indium-111 (111In, Curium)
after adding a twofold volume of 2-(N-morpholino)ethanesulfonic
acid (0.5 M MES, pH 5.5) buffer for 30 min at RT. Radiolabeling efficiency
exceeded 95% in all experiments, as determined by instant thin-layer
chromatography on silica gel chromatography strips (ITLC-SG, Agilent
Technologies) in 0.1 M sodium citrate buffer (Sigma Aldrich).
4
Ovariectomy and STZ-Induced Diabetes Model
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All mice were anaesthetized with pentobarbital sodium (50 mg/kg body weight, ip) and then ovariectomized. An incision was made along the abdominal mid-line, the ovaries were removed from both sides, and then the wound was sutured. After recovering for one week, all animals were injected intraperitoneally with STZ in 0.1 mmol/L sodium citrate buffer, pH 4.5 (Sigma Chemical Co., St. Louis, MO, USA) at a dose of 50 mg/kg/day for 5 consecutive days to induce pancreatic islet cell destruction and persistent hyperglycemia as described previously [22 (link), 23 (link)]. Some of the ovariectomized mice were administered resveratrol or 17β-estradiol (both dissolved in sesame oil, Sigma Chemical Co.) subcutaneously on every other day for 4 weeks, meanwhile at the beginning 5 days, STZ was simultaneously injected at a dose of 50 mg/kg/day intraperitoneally. Blood glucose levels were measured from the caudal vein using a portable glucose measuring device (Roche) once a week. Mice with hyperglycemia (blood glucose levels ≥300 mg/dl) were defined as a diabetic model, as before [23 (link)].
All animal procedures carried out in this study were reviewed, approved, and supervised by the Institutional Animal Care and Use Committee of the Ethics Committee of Lanzhou University, China.
All animal procedures carried out in this study were reviewed, approved, and supervised by the Institutional Animal Care and Use Committee of the Ethics Committee of Lanzhou University, China.
5
Troxerutin Ameliorates Diabetic Complications
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All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals. Forty male Sprague-Dawley rats (age 6–8 weeks, from Beijing Vital River Laboratory Animal Technology Co., certificate number 1100195543) were randomly divided into a normal control group (NC group, n = 10 rats) and a diabetic group (n = 30 rats). The rats fasted overnight. Then, STZ was dissolved in 0.1 mol/L sodium citrate buffer, pH 4.4, and the 30 rats were intraperitoneally injected with STZ citrate buffer (60 mg/kg/d, Sigma Company, USA). At 72 hours after the injection, the blood glucose level of the rats was determined via their tail veins using a blood glucose meter. Fasted rats with a blood glucose level above 16.7 mmol/L were considered to be diabetic and were randomly divided between a diabetic control group (DC group, n = 15) and a diabetic troxerutin intervention group (DT group, n = 15).
Rats in the DT group were intraperitoneally injected with troxerutin (60 mg/kg, 1 mL/kg, Jingchun Biochemistry Technology Corporation, Shanghai, China), while those in the DC and NC groups were intraperitoneally injected with physiological saline once daily for 12 weeks. Finally, the blood glucose levels were measured after 12 weeks of troxerutin treatment.
Rats in the DT group were intraperitoneally injected with troxerutin (60 mg/kg, 1 mL/kg, Jingchun Biochemistry Technology Corporation, Shanghai, China), while those in the DC and NC groups were intraperitoneally injected with physiological saline once daily for 12 weeks. Finally, the blood glucose levels were measured after 12 weeks of troxerutin treatment.
6
Chemical Preparation and Sourcing
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Chemicals including absolute ethanol, sulfuric acid, methanol, fructose,
dilute ammonium hydroxide, concentrated ammonium hydroxide, streptozotocin
(STZ), 10% formalin, phosphate buffer, and sodium citrate buffer were
purchased from Sigma-Aldrich, Germany, and all chemicals were used
without further purification. Enzyme kits were obtained from the Randox
Laboratory (Crumlin, United Kingdom).
dilute ammonium hydroxide, concentrated ammonium hydroxide, streptozotocin
(STZ), 10% formalin, phosphate buffer, and sodium citrate buffer were
purchased from Sigma-Aldrich, Germany, and all chemicals were used
without further purification. Enzyme kits were obtained from the Randox
Laboratory (Crumlin, United Kingdom).
7
Immunofluorescent Detection of Desmoglein-1
8
Immunohistochemistry of Advanced Glycation End-Products
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BP samples are from the ex vivo serum incubation study; rat subcutaneous explants were fixed in formalin (10%, v:v) for 24 to 72 h and embedded in paraffin. Five-µm-thick sections were cut via microtome and mounted on high-adherence glass microscope slides. Antigen retrieval was accomplished by 30 min incubation in 1× sodium citrate buffer (Sigma-Aldrich) at boiling temperature. Anti-AGE (Abcam; #ab23722, 1:5,000 dilution), anti-CML (Abcam #ab27684, 1:1,000 dilution), anti-HSA (Abcam #ab10241, 1:2,000 dilution), and anti-osteopontin (Abcam #ab8448, 1:2,000 dilution) were diluted in DAKO primary antibody diluent (Agilent Technologies, Santa Clara, CA, USA) and applied to sections overnight (16 to 18 h) at 4°C under gentle orbital shaking. DAKO HRP polymer-conjugated anti-rabbit secondary antibodies (Agilent, #ab214880) were applied at room temperature for 1 h. Washes were accomplished using DAKO Wash Buffer (Agilent). IHC stains were developed for 8 min using 3,3′-diaminobenzidine tetrahydrochloride substrate (Abcam), and sections were counterstained for 3 min with Gill’s hematoxylin (Sigma-Aldrich). Slides were mounted using Permount mounting medium (Fisher Scientific) and visualized by 5× light microscopy.
9
Quantification of Cell Apoptosis by TUNEL
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Cell apoptosis was evaluated by terminal deoxy-nucleotidyl transferase dUTP nick-end labeling (TUNEL), as previously described [57 (link)]. Briefly, permeabilized cells were incubated in terminal deoxy-nucleotidyl transferase buffer (0.25 U/μl terminal transferase, 6 μM biotinylated dUTP, pH 7.5; all from Roche Farma, S.A, Madrid, Spain) for 1 hour 30 minutes at 37°C in a humidified chamber. The enzymatic reaction was stopped by 15 minutes of incubation in 300 mM NaCl (Sigma-Aldrich) and 30 mM sodium citrate buffer (Sigma-Aldrich). Following an additional rinse in PBS, cultures were incubated for 30 minutes at room temperature with the avidin-biotin-peroxidase complex (1:100; Vector Laboratories, Burlingame, CA). Peroxidase activity was revealed by the 3,3′-diaminobenzidine chromogen (0.025%; Sigma-Aldrich) intensified with 0.08% NiCl2 in 30 mM Tris-HCl (pH 7.6) buffer containing 0.003% H2O2. The cell preparations were then dehydrated in ethanol (70%, 2 minutes; 80%, 2 minutes; 90%, 2 minutes; 95%, 2 minutes; 100%, 2 minutes), cleared in xylene (3 minutes), and mounted using DEPEX mounting medium (Sigma-Aldrich). Photomicrographs of TUNEL were recorded using a digital camera (Axiocam HRC; Carl Zeiss) adapted to an Axioskop 2 Plus fluorescent microscope (Carl Zeiss). For each well, 8 random fields were photographed and positive cells were scored.
10
Glycyrrhizic Acid Alleviates Diabetic Retinopathy
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All procedures with animals were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for use of animals in ophthalmic and vision research and they were approved by the institutional animal care and use committee of the College of Pharmacy, King Saud University. Adult male Sprague Dawley rats 7–10 weeks of age (200−220 g) were overnight fasted and a single bolus dose of streptozotocin (STZ) 55 mg/kg in 10 mM sodium citrate buffer, pH 4.5 (Sigma, St. Louis, MO), was injected intraperitoneally. Equal volumes of citrate buffer were injected in age-matched control rats. Rats were considered diabetic if their blood glucose was greater than 250 mg/dl.
Diabetic rats were divided into two groups: the rats in group I received normal drinking water without any supplementation and those in group II (curative treatment scheme, after disease induction) received drinking water supplemented with the potent HMGB1 inhibitor glycyrrhizic acid [52 (link)] (150 mg/kg/day, Santa Cruz Biotechnology Inc., Santa Cruz, CA) immediately after the establishment of diabetes [32 (link)-35 (link)]. After fou r weeks of diabetes, the rats were euthanized by an overdose of chloral hydrate, the eyes were removed, and the retinas were isolated and frozen immediately in liquid nitrogen and stored at -80 °C until analyzed.
Diabetic rats were divided into two groups: the rats in group I received normal drinking water without any supplementation and those in group II (curative treatment scheme, after disease induction) received drinking water supplemented with the potent HMGB1 inhibitor glycyrrhizic acid [52 (link)] (150 mg/kg/day, Santa Cruz Biotechnology Inc., Santa Cruz, CA) immediately after the establishment of diabetes [32 (link)-35 (link)]. After fou r weeks of diabetes, the rats were euthanized by an overdose of chloral hydrate, the eyes were removed, and the retinas were isolated and frozen immediately in liquid nitrogen and stored at -80 °C until analyzed.
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