The ImmunoStar neuronal nitric oxide synthase C-terminal antiserum was quality control tested using standard immunohistochemical methods. The antiserum demonstrates strongly positive labeling of rat hypothalamus, striatum, cortex and spinal cord using indirect immunofluorescent and biotin/avidin-HRP techniques. Recommended primary dilution is 1/8,000 – 1/12,000 in PBS/0.3% Triton X-100 – Bn/Av-HRP Technique.
By Western blot analysis of brain homogenates the antibody specifically labels a band of approximately 155 kD. Immuno-labeling is completely abolished by pre-adsorption with synthetic human nNOS (1419-1433) at 5 µg per mL of diluted antibody. No cross reactivity with other forms of NOS were observed.
Photo Description: IHC image of a single neuron staining for the C-terminal of nNOS in the rat cortex (above), along with low magnification of cortex (below left), neurons in the rat dentate gyrus (below middle) and in the striatum (below right). The tissue was fixed with 4% formaldehyde in phosphate buffer, before being removed and prepared for vibratome sectioning. Floating sections were incubated at RT in 10% goat serum in PBS, before standard IHC procedure. Primary antibody was incubated at 1:10000 for 48 hours, goat anti-rabbit secondary was subsequently added for 1 hour after washing with PBS. Light microscopy staining was achieved with standard biotin-streptavidin/HRP procedure and DAB chromogen, below middle image with nickel stain.
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A go to antibody for PGC spinal motor neurons
My lab has used this antibody for many years to distinguish preganglionic motor neurons in the embryonic mouse spinal cord. It is very clean and concentrated. We generally use at a 1:10,000 dilution. An example can be found in one of our papers doi: 10.1016/j.neuron.2008.06.025 (see Fig 1U).
We process our spinal cord tissues by immersion in cold 4% PFA/PBS for 1.5-2 hours depending on the size/age of the sample, wash 3X with cold PBS, and cryoprotect in 30% sucrose before cryosectioning.
Antibody staining is achieved by thawing slides, overlaying with blocking solution (PBS, 0.1% TX100 , 1% Horse (or Donkey) serum) for 15 minutes. Then add primary antibodies diluted in the blocking solution. Incubate overnight at 4 degrees. Next day wash 3X with PBST (PBS with 0.1% TX100), and add fluorescent secondary antibodies (we generally use those from Jackson Immunoresearch) for ~1-2 hours at room temp. Wash 3X with PBST and coverslip using Prolong Gold (Invitrogen).
We have used this antibody to stain for nNOS positive neurons (also stains some processes) in frozen tissue sections and whole mount preparations of mouse intestine. We get consistent staining at 1:1500 although the staining of the neurons is so strong this can probably be dropped, even for quantification purposes!
Tissue prep: 4% PFA/PBS for 1.5 hrs, cryoprotected in 30% sucrose until they sink, OCT embedded, and sectioned. For whole mounts, we open up intestine and fix in 4% PFA/PBS for 1.5 hrs.
IF protocol: Sections: Rehydrate in PBS, block with HINGS, stain in primary @4C O/N. Wmts: Wash in PBST, stain in primary with HINGS and DMSO @4C 48 hrs.
Sections: Wash with PBS, stain with Alexafluor secondaries (Invitrogen), wash with PBS, and DAPI mount. Wmts: Wash in PBST, stain in secondary with HINGS and DMSO @RT 48 hrs. Optional: proceed with MeOH dehydration and optical clearing.
Meenakshi Rao, MD PhD Assistant Professor of Pediatrics Columbia University Medical Center New York, NY 10032