The experimental procedure is shown in Figure 1 A. To induce oral tolerance, mice (7–9-week-old) were freely given sterile distilled water containing 4 mg/mL of ovalbumin (OVA; chicken albumin, Grade V; Sigma-Aldrich (St. Louis, MO, USA)) as drinking water for 7 consecutive days (oral treatment (OT) group). Control animals were given sterile distilled water (control group). On days 14 and 28, mice were immunized intraperitoneally (i.p.) with 100 μg OVA absorbed in 1 mg of aluminum hydroxide gel (Thermo Fisher Scientific, Waltham, MA, USA). On days 33 and 35, mice were challenged by intragastric gavage (i.g.) with 40 mg OVA in 200 μL of PBS using tubing. Thirty minutes after the last OVA challenge on day 35, food allergy was assessed by a decrease in rectal temperature and a diarrhea score determined on the following scale: 0: normal (normal stool), 1: minimal (soft stool), 2: slight (slightly wet and soft stool), 3: moderate (wet and unformed stool with moderate perianal staining of the coat), and 4: severe (watery stool with severe perianal staining of the coat).
Aluminum hydroxide gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
Aluminum hydroxide gel is a laboratory product used as a chemical reagent. It serves as a precipitating agent and adsorbent in various analytical and purification processes. The core function of aluminum hydroxide gel is to facilitate the separation, extraction, and concentration of target analytes or compounds from complex matrices.
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Lab products found in correlation
Ovalbumin (ova), by Merck Group (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cd4 monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Foxp3 monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Smad7 antibody, by Abcam (1 mentions)
Tgf β1 antibody, by Abcam (1 mentions)
Balb c mice, by Merck Group (1 mentions)
Female balb c mice, by Orient Bio (1 mentions)
Ova grade 5, by Merck Group (1 mentions)
Antibodies against phospho stat3 tyr 705, by Cell Signaling Technology (1 mentions)
Diaminobenzidine dab, by Solarbio (1 mentions)
Hematoxylin, by Solarbio (1 mentions)
Supersignal west femto stable peroxide substrate, by Thermo Fisher Scientific (1 mentions)
Il 17a, by MultiSciences Biotech (1 mentions)
Alexa fluor 594 labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by MultiSciences Biotech (1 mentions)
10 protocols using aluminum hydroxide gel
1
Oral Tolerance Induction in Mice
2
Murine Model of Ovalbumin-Induced Allergy
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After 4 weeks of IF, mice were sensitized by intraperitoneal injection of 50 μg OVA (Sigma-Aldrich) mixed with aluminum hydroxide gel (Thermo Fisher Scientific) on days 28 and 35. From day 42 to 49, mice were challenged by intragastric administration of 10 mg OVA dissolved in 0.2 mL PBS every other day. On the last day of the protocol (day 56), mice were challenged by intragastric infusion of 50 mg OVA (48 (link)) (Figure 1A
3
Experimental Protocol for Allergic Asthma
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For the detailed experimental protocol, we followed the methods of Jang et al, 2016.[3 (link)] For the induction of allergic asthma, the mice were first sensitized with an intraperitoneal (i.p.) injection of 20 μg of OVA (Sigma-Aldrich, St. Louis, MO, USA) and 2.25 mg aluminum hydroxide gel (alum adjuvant; Thermo Fisher Scientific, Waltham, MA, USA) in 100 μl of sterile saline on days 0 and 14. After systemic sensitization, the mice were locally challenged by intranasal (i.n.) instillation of 500 μg of OVA into their nostrils from days 28, 29, and 30.
4
Modulation of T-cell Activation Markers
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Ovalbumin, PMA, and 1A,245-dihydroxyvitamin D3 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Aluminum hydroxide gel, FOXP3 monoclonal antibody, and IL-17 monoclonal antibody were purchased from Thermo Fisher Scientific (Shanghai, China). CD4 monoclonal antibody and CD 25 monoclonal antibody were purchased from Invitrogen (Carlsbad, CA, USA).
5
Inhalation-based Immunomodulation for Asthma
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Ovalbumin (OVA) was obtained from Sigma-Aldrich (St. Louis, MO), aluminum hydroxide gel from Thermo Fisher Scientific (Waltham, MA), and M. vaccae injections from Anhui Chi dragon coma Biological Pharmaceutical Co. The general SP detection kit and the 3,3′-diaminobenzidine tetrahydrochloride (DAB) chromogenic reagent kit were obtained from Beijing Jinqiao Biological Technology Co., Ltd. (Beijing, China). TGF-β1 antibody, TβR1 antibody, Smad1 antibody, and Smad7 antibody were obtained from Abcam (Cambridge, United Kingdom).
Other materials and equipment used were type IV collagenase, mouse viscera lymphocyte separation solution (Haoyang, Tianjing, China), phorbol 12-myristate 13-acetate (PMA)/lonomycin mixture, fetal bovine serum, FIX&PERM Kit, PE-CY5-anti-mouse CD3, FITC-IgG1, FITC-anti-mouseγδTCR, PE-IgG1, PE-anti-mouse-IL-13, PE-anti-mouse-LAP, glycogen staining kit, Wright stain, hematoxylin–eosin (HE) staining kit, cytometer, electron microscope, high-speed cryogenic centrifuge, ultrasonic atomizer WH-2000 (Yuehua medical instrument factory, Guangdong, China), atomized inhalation box (self-made), carbon dioxide incubator, lung function machine (Buxco, USA), 10% chloral hydrate, normal saline, 10% formaldehyde, and phosphate-buffered solution (PBS).
Other materials and equipment used were type IV collagenase, mouse viscera lymphocyte separation solution (Haoyang, Tianjing, China), phorbol 12-myristate 13-acetate (PMA)/lonomycin mixture, fetal bovine serum, FIX&PERM Kit, PE-CY5-anti-mouse CD3, FITC-IgG1, FITC-anti-mouseγδTCR, PE-IgG1, PE-anti-mouse-IL-13, PE-anti-mouse-LAP, glycogen staining kit, Wright stain, hematoxylin–eosin (HE) staining kit, cytometer, electron microscope, high-speed cryogenic centrifuge, ultrasonic atomizer WH-2000 (Yuehua medical instrument factory, Guangdong, China), atomized inhalation box (self-made), carbon dioxide incubator, lung function machine (Buxco, USA), 10% chloral hydrate, normal saline, 10% formaldehyde, and phosphate-buffered solution (PBS).
6
Glucosamine Alleviates Atopic Dermatitis in Mice
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This study was carried out in female BALB/c mice (Orient Bio Inc., Seongnam, Korea) aged 8 weeks with AD induced by ovalbumin (OVA; Sigma-Aldrich Korea, Yongin, Korea). Twenty-five BALB/c mice were divided into five groups of five mice: control group without induction of AD (group A), AD group with null treatment by phosphate-buffered saline (PBS) (group B), and AD groups treated with 10 mg, 20 mg, and 40 mg of GlcN (Sigma-Aldrich Korea) administration (groups C, D, and E, respectively). For the AD induction groups (groups B~E), 1.5 ml of OVA and 3 ml of aluminum hydroxide gel (Thermo Fisher Scientific, Waltham, MA, USA) were mixed, and 150 µl of the mixture was intraperitoneally injected into the mice three times a week for three weeks. After a week of OVA injection, mice were epicutaneously sensitized with OVA patches. The patches were made by dripping 50 µl of OVA (1 mg/ml) in a gauze (1×1 cm) which was placed on the back skin three times per week for two weeks. Group A was injected with PBS instead of OVA. This experiment was conducted in specific-pathogen-free environment and the mice received an OVA-free diet. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Inha University (INHA 181120-601).
7
Allergic Airway Inflammation Induction and Treatment
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Allergic airway inflammation was induced as previously described with minor modifications (22 (link)). Eight-week-old mice were sensitized using intraperitoneal (i.p.) injection of 100 μg OVA (grade V) emulsified in 1 mg of aluminum hydroxide gel (Thermo Scientific, Rockford, IL) in a total volume of 200 μl on days 0 and 7, respectively. Mice injected with an equal volume of saline served as controls. The sensitized mice were anesthetized with diethyl ether and challenged by i.t. administration of 50 μl OVA (20 μg/μl) or saline for 3 consecutive days on days 11, 12 and 13. Mice were euthanized at day 14. Lungs and BALF were harvested for histological studies, differential cell counting and biochemical analyses. For the therapeutic experiment, one day before OVA challenge (day 10), the mice were administered with scrambled or Mbd2 siRNA-loaded liposomes by i.t. injection (1 mg/kg) with diethyl ether anesthesia, and the other procedures were the same as those described above (Figure 5B).
8
Inflammatory Cytokine Quantification Protocol
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OVA (grade V) was obtained from Sigma (USA), aluminum hydroxide gel was obtained from Thermo (USA) and anti-rat IL-6/IL-1β/TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Feiya Biotech (China). Baicalein was provided by Dr Qinglong Guo of the College of Pharmacy at China Pharmaceutical University. Antibodies against phospho-STAT3 (Tyr705) were obtained from Cell Signaling Technology (USA), which were used for immunohistochemical staining at 1:100 dilution. BCA Protein Assay kit was purchased from Thermo Fisher Scientific (USA). The kit for immunohistochemical staining was obtained from Keygen biotechnology Co., Ltd. (China). Diaminobenzidine (DAB) was obtained from Solarbio Science & Technology Co., Ltd. (China). Hematoxylin and eosin were purchased from Solarbio Science & Technology Co., Ltd. (China).
9
Andrographolide Modulates Immune Responses
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Andrographolide (C20H30O5) was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). The sample consisted of a white powder with a molecular weight of 350.45 and a purity of above 98% determined by HPLC analysis. OVA was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). The aluminum hydroxide gel was purchased from Thermo Fisher Scientific (Watham, MA, USA). The IL-6, IL-17A, and IL-17F ELISA kits were purchased from Multi Sciences (Hangzhou, China). Primary antibodies and HRP-conjugated anti-rabbit IgG were purchased from Bioworld Technology (Minnesota, USA), Cell Signaling Technology (Beverly, MA, USA), Affinity Bioscience (Shanghai, China), and Proteintech Group (Wuhan, China), respectively. SuperSignal West Femto Stable Peroxide Substrate and AlexaFluor594-labeled goat anti-rabbit IgG antibodies were obtained from Thermo Scientific (San Jose, CA, USA).
10
Allergic Asthma Induction and Probiotic Treatment
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The mice in the control group were sensitized and challenged with normal saline. For the induction of allergic asthma, mice in the OVA group were sensitized with an intraperitoneal (i.p.) injection of 20 μg OVA (Sigma-Aldrich, St. Louis, MO, USA) and 2.25 mg aluminum hydroxide gel (as an alum adjuvant: Thermo, Waltham, MA, USA) in 100 μL saline on days 0 and 14. After systemic sensitization, mice were challenged by intranasal instillation of OVA (500 μg) on days 28, 29, and 30 (Fig. 1).
LP was obtained from the Korean Collection of Type We diluted LP or BC in normal saline and administered it orally, every day from the week before the first sensitization to the last OVA challenge. Mice in the LP1 or BC1 group were administered 0.2 mL of LP or BC at doses of 1×10 9 . CFU/mL. Mice in the LP5 or BC5 group were administered 0.2 mL of LP or BC, at doses of 5×10 9 CFU/mL, respectively (Fig. 1).
LP was obtained from the Korean Collection of Type We diluted LP or BC in normal saline and administered it orally, every day from the week before the first sensitization to the last OVA challenge. Mice in the LP1 or BC1 group were administered 0.2 mL of LP or BC at doses of 1×10 9 . CFU/mL. Mice in the LP5 or BC5 group were administered 0.2 mL of LP or BC, at doses of 5×10 9 CFU/mL, respectively (Fig. 1).
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