Blood samples of clean mice were collected before immunization. The blood sample was incubated for 1 h at room temperature and then centrifuged at 3,913 × g for 15 min at 4°C. The serum was collected as a negative control. Mice were immunized by abdomen multipoint subcutaneous injection using 30 μg of OmpA dissolved in 100 μL of PBS, which was mixed with an equal volume of Freund's complete adjuvant (Gibco). After 2 wk, the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant (Gibco) and the mice were immunized in the same way. A total of 4 immunizations were performed. After the third immunization, blood was taken from the tail vein for ELISA verification. The last immunization was a booster immunization, and 30 μg of pure protein diluted with PBS was injected intraperitoneally. Subsequently, whole blood was collected 3 d later after the last immunization from the eye socket; the serum was collected according to the above described. The polyclonal antibody specific for OmpA was produced. We then added glycerin to the antibody at a ratio of 1:1, measured the concentration, and stored it at -40°C. Animal experiments and procedures were implemented in accordance with the guiding principles of animal experiments and approved by the Experimental Animal Committee of Kunming University of Science and Technology.
Complete freund s adjuvant
Manufactured by Thermo Fisher Scientific
Sourced in United States
Complete Freund's adjuvant is a laboratory reagent used to enhance the immune response in experimental animals. It is a water-in-oil emulsion containing killed and dried mycobacteria. The primary function of Complete Freund's adjuvant is to stimulate the immune system, although its specific applications should be determined by the user.
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Lab products found in correlation
Freund s incomplete adjuvant, by Thermo Fisher Scientific (16 mentions)
Urea, by Xilong (3 mentions)
Percoll, by GE Healthcare (2 mentions)
Methylated bovine serum albumin (mbsa), by Merck Group (2 mentions)
Mycobacterium tuberculosis h37ra, by BD (2 mentions)
Bovine type 2 collagen, by Chondrex (1 mentions)
Pertussis toxin, by Thermo Fisher Scientific (1 mentions)
Mog35 55, by Sangon (1 mentions)
Aluminum hydroxide gel, by InvivoGen (1 mentions)
Luciferase assay reagent, by Promega (1 mentions)
Ovalbumin (ova), by InvivoGen (1 mentions)
Cpg odn, by Thermo Fisher Scientific (1 mentions)
Plasmid extraction kit, by Tiangen Biotech (1 mentions)
Sybr color qpcr master mix, by Vazyme (1 mentions)
Ecori, by Takara Bio (1 mentions)
Goat anti mouse igg hrp, by Proteintech (1 mentions)
Rna extraction kit, by Vazyme (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
Bamhi, by Takara Bio (1 mentions)
41 protocols using complete freund s adjuvant
1
Polyclonal Antibody Production against OmpA
2
Inducing Arthritis in DBA/1 Mice
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Arthritis was induced in 9-week-old DBA/1 mice in accordance with the Ethical Committee for animal experimentation of the Languedoc-Roussillon (Approval APAFIS#5347-2016050917427820). Briefly, bCII (2 mg/mL) was diluted in acetic acid (0.05 M) and emulsified in Freund´s complete adjuvant (Thermoscientific, Rockford, IL, USA) as described 10 (link). The suspension (100 µL) was injected intradermally at the base of the tail at day 0. At day 21, a boost with bCII in Freund´s incomplete adjuvant was administered. EVs were injected intravenously at day 18 and 24. Clinical signs of arthritis were scored as reported 19 (link). At euthanasia, blood, draining lymph nodes and spleens were collected for immune cell analysis. Hind limbs were fixed in 4% formaldehyde for X-ray micro-computed tomography (µCT).
3
Collagen-Induced Arthritis Model in Mice
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Arthritis was induced, as previously described [54 (link)]. Bovine type II collagen (2 mg/mL) (Chondrex Inc. Redmond, WA, USA) was emulsified in equal volumes of Freund’s complete adjuvant (Thermofisher Scientific Inc. Waltham, MA, USA). On day 0, the mice were immunized by subcutaneous (s.c.) injection of the emulsion (100 μL) at the base of the tail. On day 21, animals received an intraperitoneal booster injection of collagen II (2 mg/mL, 100 μL) that was dissolved in phosphate buffered saline (PBS).
4
Chicken IL-10 Antibody Production
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Single Comb White Leghorn laying hens raised for life in cages with raised wire were injected (100 μg of conjugate per chicken) with bovine IL-10-bovine gamma globulin-vmpqaenh conjugate emulsified with Freund's Complete Adjuvant at 1:1 vol/vol (Thermo Fisher Scientific Inc., Waltham, MA) for a total volume of 1 mL per chicken. The protein sequence VMPQAENH was used to conjugate to the carrier protein bovine gamma globulin. Sequence VMPQAENH is the portion of the IL-10 sequence that was used to make the antibody to IL-10 for use in dairy calves. This protein sequence was made at GenScript (Piscataway, NJ). Hens injected with adjuvant and bovine gamma globulin only (no peptide conjugate) were used for making control antibodies. Chickens were injected 1 wk later using the antigens described above. Eggs were collected beginning 3 wk after the initial injection for a period of 8 wk. Egg yolks were separated from the albumen, lyophilized, and stored at room temperature until needed. Enzyme-linked immunosorbent assays were then used to determine the titer of the antibody.
5
Induction and Evaluation of Experimental Autoimmune Encephalomyelitis
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The EAE mouse model was induced as described previously (61 (link)). Briefly, female mice at the age of 12 weeks were immunized with 300 mg of MOG35–55 (Sangon) in Freund’s Complete Adjuvant (Thermo Fisher Scientific) and treated with 200 ng of pertussis toxin (Gibco) intraperitoneally on days 0 and 2.
For T cell adoptive transfer, CD4+ T cells were collected from WT and lncRNA-GM−/− mice and then intravenously transferred into Rag2−/− mice at 5 × 106 per mouse. The recipient Rag2−/− mice were immunized with MOG35–55 and treated with pertussis toxin at days 0 and 2. Clinical scores were evaluated according to the following standards: 0, normal; 1, limp tail; 2, limp tail, impaired righting reflex, and paresis of one limb; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; and 5, moribund.
For flow cytometric analysis of brain and spinal cord mononuclear cells, single-cell suspensions from brain and spinal cord were poured into 35% Percoll (GE Healthcare) reagent. The cells at the bottom layer were then collected and used immediately for antibody staining.
For T cell adoptive transfer, CD4+ T cells were collected from WT and lncRNA-GM−/− mice and then intravenously transferred into Rag2−/− mice at 5 × 106 per mouse. The recipient Rag2−/− mice were immunized with MOG35–55 and treated with pertussis toxin at days 0 and 2. Clinical scores were evaluated according to the following standards: 0, normal; 1, limp tail; 2, limp tail, impaired righting reflex, and paresis of one limb; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; and 5, moribund.
For flow cytometric analysis of brain and spinal cord mononuclear cells, single-cell suspensions from brain and spinal cord were poured into 35% Percoll (GE Healthcare) reagent. The cells at the bottom layer were then collected and used immediately for antibody staining.
6
Chicken Antibody Generation against Recombinant PGRN
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Recombinant human PGRN was obtained from Sino Biologicals. For the initial immunization, 200 ug of protein was emulsified in 250uL of Freund’s Complete Adjuvant (Thermo Fisher, 77140) and injected into the breast tissue. Fourteen days later, chickens were boosted with 100 ug protein and 250uL of Freund’s Incomplete Adjuvant (Thermo Fisher,77145). Animals were subsequently boosted every other week, with blood drawn on the off-week to assess antigen-specific titer. Four days before euthanization and spleen harvesting, animals received a final IV boost with no adjuvant.
7
Antibody Response in BALB/c Mice
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The mice were handled in accordance with the requirements of the Institutional Animal Ethics Committee (IAEC) of Jawaharlal Nehru University and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Three female BALB/c mice (6-weeks old) were immunized intraperitoneally (IP) with 30 μg of recombinant protein (per mouse; in 0.9% saline) mixed with an equal volume of Freund’s complete adjuvant (ThermoFisher Scientific) on day 0, followed by 2 booster regimens in Freund’s incomplete adjuvant (ThermoFisher Scientific) on days 21 and 42. Bleeds were collected from each mouse 10 days following each booster dosage. The antisera obtained from the terminal bleeds of the mice were used for all the experiments, at a dilution of 1:250.
8
Equine Immunization Protocol for RBD Protein
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Three 5~6-year-old healthy male horses were fed under standard conditions. After emulsifying 1.5 mg of RBD protein in 1 mL PBS with an equal volume of Freund’s complete adjuvant (Thermo Fisher, USA), horses were vaccinated intramuscularly via a subcutaneous multipoint injection route. The subsequent immunization dosage was, respectively given 2.5, 2.5, 3.0, and 4.0 mg of protein in 1 mL PBS mixed with equal volume Freund’s incomplete adjuvant (Thermo Fisher, USA) for each horse via subcutaneous multipoint injection route. Each horse was immunized 5 times in total at 14-day intervals (Figure S2A
9
CpG-ODN Adjuvant Immunization Protocol
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CpG-ODN were purchased from Invitrogen or Genomics Biosci and Tech. Ovalbumin and aluminum hydroxide gel were purchased from Invivogen. Freund’s complete adjuvant and incomplete adjuvant were purchased from Thermo Scientific. Luciferase assay reagents were purchased from Promega.
10
Polyclonal Anti-Trichomonas vaginalis Antibody
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The production of the polyclonal anti-T. vaginalis antibody followed a protocol previously described [20 (link)] with some modifications. Briefly, 1 × 107 trophozoites of T. vaginalis (VPFS strain) were concentrated by centrifugation. The pellet was resuspended in 1 mL PBS (pH 7.2) and sonicated (40 Hz frequency) for 1 min with three repetitions. After that, two Wistar, 10-week-old female rats weighing approximately 270 g were subcutaneously inoculated with 0.5 mL of sonicate emulsified in Freund's complete adjuvant (Thermo Fisher Scientific, USA). Fifteen days after the first inoculation, the animals received a booster with the material obtained as described for the first immunization without the adjuvant. Ten days after, these animals were exsanguinated through the ribcage for blood collection. Subsequently, the blood was centrifuged in order to obtain the anti-T. vaginalis antibody and kept at −20°C until the immunocytochemical reaction was carried out.
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