Cryosections were rinsed in PBS and baked at 60°C for 45 min. Slides were then fixed in 4% PFA for 1 hour at room temperature. Ethanol dehydration was then performed with 50%, 70%, 100%, 100% ethanol for 5 minutes each. After air drying, sections were treated with RNAScope® hydrogen peroxide for 10 minutes, followed by antigen retrieval at 95°C in RNAScope® Target Retrieval Buffer. Slides were then baked at 60°C for 45 min. The remaining steps of the in situ hybridization procedure were performed using the RNAScope® Multiplex Fluorescent Reagent Kit v2 according to manufacturer guidelines. Upon completion of the in situ, CTb immunostaining was performed. Briefly, spinal cord sections were blocked in PBS containing 0.2% Triton-X and 10% donkey serum (Jackson ImmunoResearch). Rabbit anti-CTb (ab34992, Abcam) diluted at 1:8,000 in PBS containing 0.2% Triton-X was incubated overnight at 4°C. The following day, secondary antibody labeling was performed with donkey anti-rabbit-Alexa488 (Invitrogen).
Ab34992
Manufactured by Abcam
Sourced in United Kingdom
Ab34992 is a laboratory instrument used for the detection and quantification of specific proteins or other biomolecules in a sample. The core function of this product is to provide a reliable and efficient means of conducting immunoassays, a common technique in biochemical and biomedical research.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti rabbit alexa 488, by Thermo Fisher Scientific (2 mentions)
Donkey serum, by Jackson ImmunoResearch (2 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Gtx213110 01, by GeneTex (1 mentions)
Monosialoganglioside gm1, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
5 protocols using ab34992
1
Multiplex Fluorescent In Situ Hybridization and CTb Immunostaining
2
Quantitative ELISA for Cholera Toxin B
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The wells of transparent 96-well microtiter plates were coated with 100 μl of mouse anti-CTB (Abcam 35988, diluted 1: 1,000 in PBS) for 16 h at 4°C. The wells were then washed with 1 × PBST three times and blocked with blocking buffer (1% BSA in 1 × PBS) for 1.5 h. Soluble cytoplasmic fractions prepared as described above (100 μl) were added to each well and incubated for 2 h. The samples were removed, and the wells were washed three times; then, 100 μl of the primary antibody (rabbit anti-CTB, Abcam ab34992) 1/2,000 diluted in 1 × PBS were added. After 1 h of incubation, the primary antibody was removed, and the wells were washed three times. Then the secondary antibody (goat anti-rabbit IgG (HRP), GeneTex GTX213110-01) 1/5,000 diluted in 1 × PBS was added. The secondary antibody was removed, and the wells were washed three times, and then TMB solution was added. After adding the stop solution, we measured the samples using a plate reader (TECAN, Infinite 200 PRO) at O.D.450.
3
Cholera Toxin Antibody Procurement
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CT and monosialoganglioside GM1 were purchased from Sigma-Aldrich® (St. Louis, MO, USA). Rabbit polyclonal antibody against CT (ab123129) and CTB (ab34992) were obtained from Abcam (Cambridge, UK). Rabbit polyclonal antibody against CTA (AB-43) was purchased from Advanced Targeting Systems (San Diego, CA, USA).
4
Western Blot Analysis of TcpA and CTB
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The bacterial cells for the western blot analysis of TcpA or bacterial culture for the western blot analysis of CTB (approximately 107 cells/well) were analyzed using 12 or 15% SDS-PAGE. For western blotting, proteins separated by SDS-PAGE were transferred onto a nitrocellulose membrane. The membrane was blocked for one hour at room temperature in TBS blocking buffer. Primary antibodies (anti-TcpA (a generous gift from Dr. W. F. Wade, Dartmouth University, Hanover, NH, USA [46 (link)]), and anti-CTB (ab34992, Abcam, Cambridge, UK) were diluted 1:10,000) were added to the membrane and incubated overnight at 4 °C. The membrane was then washed with TBST, and a secondary antibody (goat anti-rabbit IgG (diluted 1:20,000) or goat anti-mouse IgG (diluted 1:5000)) was added. Western blot images were analyzed using an Odyssey CLx imaging system (LI-COR Biosciences, Lincoln, NE, USA). The western blot band intensities representing TcpA and CTB were quantified using the ImageJ gel analysis program and LI-COR Odyssey software.
5
Multiplex Fluorescent In Situ Hybridization and CTb Immunostaining
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