Citations

 Table 1. List of immunological reagents, source and concentration

Injection of retrograde tracers:

Following recovery (710 days) from the NTS injection, animals were re-anaesthetised (as previously described) and either 1 l of 2% fluorogold (FG) or 2 l of 0.5% cholera toxin B subunit (CTB, Table 1) was pressure injected into the left side spinal cord intermediolateralis …

Biotin dextran aminecholera toxin B subunit for epifluorescence:

BDA sections were incubated in 10% NHS0.3% Triton-X-100 for 45 min, rinsed in Tris-buffered saline (TBS, 1  10 min) and then incubated in an appropriate streptavidin Alexa Fluor 594 or 488 for 2 h. The sections then had a further rinse in TBS followed by avidinbiotin blocking and incubation in goat anti CTB with streptavidin Alexa Fluor 594 or 488 … 

• Product #103 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Transganglionic Labeling of Sciatic-Projecting Sensory Axons. Three days before sacrifice, sciatic nerves were injected bilaterally with a 1% (wt/vol) solution of CTB (List Biological Laboratories). …

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

Induction and evaluation of EAE:

Female C57BL/6 mice (8-10 week old) were immunized subcutaneously in the dorsal flank with 150 µg of myelin oligodendrocyte glycoprotein peptides (MOG₃₅₅₅) emulsified in complete Freund’s adjuvant (CFA; Chondrex, Seattle, WA, USA) on days 0 and 7. The mice were injected intraperitoneally (i.p.) with pertussis toxin (List Biological Laboratories, Campbell, CA, USA) at a dose of 500 ng per mouse on days 0 and 2, and at a dose of 200 ng per mouse on day 8. …

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)

Methods:

Recombinant GST-SV2c (List Prod #690) and recombinant GST Synaptobrevin-2 (List Prod #510A), a control protein, were attached to glutathione coated magnetic beads. Each set of coated beads was exposed to varying amounts of BoNT/A in buffer, washed, and incubated with SNAPtide (List Prod #520), a quenched fluorogenic (FRET) substrate. Detection of cleaved peptide was monitored using reverse phase HPLC with fluorescence detection.  

• Product #690B – Receptor for Clostridium botulinum Type A Neurotoxin, GST-SV2c luminal domain
• Product #510A – Recombinant GST Synaptobrevin-2 (PRODUCT DISCONTINUED)
• Product #520 –  SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin

TNA:

The titer of toxin-neutralizing antibody in serum was determined using modifications to the methods of Pitt et al [15] and Little et al [4] by testing the ability of the serum to inhibit the cytotoxicity of the combination of rPA with LF (List Biological Laboratories Inc., Campbell, CA, USA). The assay was carried by exposing J774A.1 murine macrophages (ATCC TIB-67) to PA and LF in the presence or absence of serum. In brief, J774A.1 cells were plated out on 96 well plates (SPL CO. Ltd, Pyeongtaek-si, Korea) and seeded with 10⁶ cells per well in 150 l volumes 2 hours before testing. …

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Cell Lines, Purified C. difficile Toxins, and Supernatants:

… Purified toxin and toxoid proteins … were obtained from List Biological Laboratories (Campbell, California …

Author did not specify which C. difficile toxin an toxoids were utilized; List Labs provides the following C. difficile products:  https://listlabs.com/product-information/difficile-toxins/

TC DNA immunization by applying plasmid DNA onto a mouse skin area wherein the hair was depilated by warm-waxing:

… Two days after the plucking, mice were anesthetized again, and the plucked area was cleaned with 70% ethanol, hydrated for 20 min with warm water, and paper-dried. Plasmid DNA (pCMV- or pCI-neo-sOVA, 50 µg) admixed with 10 µg cholera toxin (CT) (List Biological Laboratories, Campbell, CA) was gently dripped onto the hydrated area using a pipette tip. …

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

Cell culture:

Human neonatal epidermal melanocytes (HEM1455) were obtained from Cell Applications (San Diego, CA). Melanocytes were grown in HAM’s F10 media supplemented with ITS premix (Becton Dickinson, Franklin Lakes, NJ), 12-O-tetradecanoylphorbol-13-acetate (Sigma-Aldrich, St Louis, MO), 3-isobutyl-1-methylxanthine (Sigma-Aldrich), cholera toxin (List Biological Laboratories, Campbell, CA), …

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

Axon tracing:

Retrograde neuronal tracing was performed via injection of cholera toxin Beta-subunit (CTB) following injury. Four days prior to euthanizing, the mice were re-anesthetized, with a constant delivery of isoflurane, and the right sciatic nerve was exposed. Two microliters of a 1.5% solution of CTB (List Biological Laboratories) was injected into the nerve using a Hamilton syringe. The solution was slowly injected over a 1 min period and the syringe was withdrawn over an additional 2 min to prevent reflux. The muscle and skin were then sutured. Spinal cords were isolated, and cryosectioned at 18 m thickness. Alternating sections were stained against CTB via goat anti-CTB genotoxiod (List Biologicals) and detected with a fluorescently conjugated secondary. The ImageJ software (NIH) was used to measure neurite length. Neurite length was measured and plotted as distance from injury epicenter (0 m).

Immunohistochemistry:

Terminally anesthetized mice were perfused trans-cardially with PBS, followed by 4% paraformaldehyde in PBS (pH 7.4). Spinal cords were isolated, post fixed for 1 h and cryoprotected in 30% sucrose overnight. 18 m frozen sections were prepared, blocked for 1 h with 5% serum and then incubated with primary antibodies (rabbit anti-IBA-1 1:500, Wako; rabbit anti-GFAP 1:500, Dako; mouse anti-APC (CC1), 1:100, Millipore; rabbit anti-NG2, 1:500 a generous gift from Dr. Joel Levine; goat anti-CTB, 1:5000, List Biologicals; mouse anti-CD3, Dako) overnight at 4 C. …

Author did not specify which CTB was utilized.  List Labs provides Product #103B (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae)  and Product #104 (Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt).

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Materials:

VAMPtide substrates (Products #540, #541 and #542), BoNT/B light chain (LC ) (Product #620A), and BoNT/B holotoxin (Prod #136A) are products of List Biological Laboratories, Inc.

• Product #540 – VAMPtide Peptide Substrate (o-Abz/Dnp) Substrate for C. botulinum Type B Neurotoxin
• Product #541 – VAMPtide Peptide Substrate (FITC/DABCYL) Substrate for C. botulinum Type B Neurotoxin
• Product #542 – VAMPtide (Pya/Nop) Peptide Substrate for Botulinum Neurotoxin Type B
• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant
• Product #136A – Botulinum Neurotoxin Type B, Nicked, from Clostridium botulinum

Methods – The above materials were used in the following (refer to poster for details):

• Fluorimetric assays
• Sensitivity and LOD