Citations

EAE induction and clinical score:

… In addition, mice received 200 ng pertussis toxin (List Biological Laboratories, Hornby, ONT, Canada) by intraperitoneal injection at the day of immunization and 48 h later. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

… Each administration provided approximately 1 x 10 9 inactivated bacteria with either 7.5 µg CT (List Biological Laboratories, Inc., Campbell, CA) …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

– BM-DCs (1 3 106 cells/ml of 10% CM with GM-CSF [20 ng/ml]) were then cultured in the presence of either C. neoformans-MPs (50 mg/ml), LPS (1 mg/ml; List Biological Laboratories, Campbell, CA), or vehicle (PBS) at 37C and 5% CO2 for 24 h. …

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

… HPMVECs and HPMEC-ST1.6R cells were challenged with 1-10 g/ml LPS (Escherichia coli 055:B5; List Biological Laboratories, Campbell, CA) …

NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) may have been utilized.

Product #203 (5 mg – LPS from Escherichia coli O55:B5) is no longer sold.

Product #203A (2.5 mg – LPS from Escherichia coli O55:B5) is available for purchase.

• Product #203A – LPS from Escherichia coli O55:B5

… Plates were incubated at 37 o C in 5% CO 2 for 24 hrs. Each plate included control wells untreated and treated with purified C. difficile toxin B (List Laboratories, Campbell, CA) …

• Product #155A – Toxin B from Clostridium difficile

… For active EAE, mice were immunized subcutaneously with 200 µg mouse MOG35-55 (CS Bio) emulsified in CFA (Sigma-Aldrich) and were injected intraperitoneally with 200 ng pertussis toxin (List Biologicals) on days 0 and 2 after immunization. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Eye Injections, Perfusions and Section Collection

Animals were anesthetized with an intramuscular injection of ketamine (5 mg/Kg; Agener), xylazine (0.5 mg/kg; Rhobigarma), diazepam (0.5 mg/Kg; Compaz) and tramadol hydrochloride (5 mg/kg; Cristália). Following topical applications of tetracaine hydrochloride (Allergan) to the cornea, bats were given a unilateral eye injection (left eye) of an aqueous solution that included 15 μl of 5% of the B subunit of cholera toxin (CTb, List Biological Laboratories, Inc., Campbell, CA, USA) in 10% dimethyl sulfoxide (DMSO). This solution was injected into the vitreous humor using a 30-gauge needle catheter attached to a micropump, which pushed the solution at a rate of 0.8 μl/min. To minimize the reflux and spread of the tracer to the extraocular muscles, the needle was left on the site until 15 min post-injection and then withdrawn. To avoid post-operatory local infection, the ocular surface was cleaned with saline during the surgical procedure. Then, the ocular surface was washed with saline and an antibiotic ointment was topically applied. Five days post-injection, bats were reanesthetized with the same anesthetic and perfused transcardially with 150 ml of phosphate-buffered saline, pH 7.4, containing 500 UI heparin (Liquemine, Roche, Brazil), followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brain was removed and, after postfixation in the same fixative for 2 h, were cut serially into coronal 30 μm sections using a freezing microtome. The sections were collected individually and placed into a series of six jars filled with PB for subsequent staining. Thus, the anteroposterior interval among sections stained for CTb or thionin was 180 μm.

Nissl Staining and Immunohistochemistry

Sections from one series were immediately mounted onto electrostatic glass (Fisherbrand) and were then Nissl stained with thionin to visualize the cytoarchitectonic delimitation of the neuronal groups. Sections from another series were submitted to immunohistochemistry to reveal CTb. All of the immunohistochemistry procedures were performed at room temperature. The sections, previously submitted to pre-treatment with hydrogen peroxide (H2O2), were free floating incubated in a blocking solution containing bovine serum albumin (BSA); diluted in 5% Triton X-100 for 1 h; and incubated for 18–24 h with the primary antiserum, a goat anti-CTb IgG (List Biological Labs, Campbell, CA, USA; RRID: AB_10013220) diluted 1:1,000 in solution containing 2% BSA, 0.4% Triton X-100 and 0.1 M PB, pH 7.4. The sections were then incubated with a biotinylated secondary antiserum (donkey anti-goat IgG, JacksonLabs, Westgrove, PA, USA) diluted 1:1,000 for 90 min. The sections were subsequently incubated with an avidin–biotin–peroxidase solution (ABC Elite kit, Vector Labs, Burlingame, CA, USA) for 90 min in 0.4% Triton X-100 NaCl. The sections were then reacted for peroxidase in a solution of diaminobenzidine tetrahydrochloride (DAB, Sigma, St Louis, MO, USA) and 0.01% H2O2 in 0.1 M PB, pH 7.4. The sections were washed with 0.1 M PB, pH 7.4 (5 × 5 min) between each step and at the end of the procedure. The sections were then allowed to dry, dehydrated through a graded alcohol series, cleared in xylene, and cover-slipped with the neutral mounting medium ERV-MOUNT (EasyPath). Specificity tests were performed based on omission of the primary or secondary antibodies in some sections. In all cases immunolabeling was completely abolished. Furthermore, as a control for the possibility of transsynaptic labeling, the occipital lobes of one animal were sectioned and processed for CTb immunostaining. The visual cortex of these animals was examined to verify the presence of CTb. As a result, immunolabeling was completely abolished.

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Antigen-specific IgE:

Low abundance of serum antigen-specific IgE meant a modified, multiplexed Luminex assay (Luminex Corporation, Austin, TX) was employed to measure tetanus and acellular pertussis antigen-specific IgE responses. Sera were treated with GullSORB (Meridian Bioscience, Inc., Cincinnati, OH) to remove potentially competing IgG and incubated with purified pertussis toxin mutant (PTM), pertactin, or tetanus toxoid (all from List Biological Laboratory, Campbell, CA) coupled to fluorescent-barcoded MagPlex microspheres (Luminex Corporation). …

• Product #184 – Pertussis Toxin Mutant
• Product #187 – Pertactin from B. pertussis (69 kDa Protein)
• Product #191 – Tetanus Toxoid from Clostridium tetani

Oral Sensitization and GI Antigen Challenge:

Oral allergic sensitization and oral allergen challenge were performed as depicted in Figure 1A. For oral sensitization, mice were given 1 mg of OVA (grade V, Sigma-Aldrich, St. Louis, MO, USA) and 15 µg of CT (List Laboratories, Campbell, CA, USA) in …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Induction of EAE and CBD Treatment Regimen:

Experimental autoimmune encephalomyelitis was induced in groups of 10 female C57BL/6 mice (68 weeks old) as described previously … Following immunization, 200 ng of pertussis toxin (List Labs, Campbell, CA, USA) was injected i.p. into mice on day 0, followed by a 400 ng pertussis toxin intraperitoneally (i.p.) injection on day 2.

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)