Rehydrated identical brainstem sections were treated with 1% H2O2 to block endogenous peroxidase activity followed by citrate pretreatment as above. Sections were washed with the PBS solution and were blocked with 10% normal horse serum and incubated overnight with anti‐SMI32 (Covance, 1:500) at 4°C. Sections were incubated with biotinylated horse antimouse (1:100) for 45 min. The antigen was visualized and the sections mounted as above.
Anti smi32
Manufactured by Fortrea
Sourced in United States
Anti‐SMI32 is a laboratory reagent used in immunohistochemical and immunofluorescence applications. It functions as a marker for non-phosphorylated neurofilament H, which is present in healthy and degenerating neuronal axons.
Automatically generated - may contain errors
Lab products found in correlation
Anti tom20, by Merck Group (1 mentions)
Alexa flour 594, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa flour 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anticholine acetyltransferase, by Abcam (1 mentions)
Anti cd11b, by Bio-Rad (1 mentions)
Anti gp91phox, by BD (1 mentions)
Anti arginase 1 arg1, by Abcam (1 mentions)
Anti cd163, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Santa Cruz Biotechnology (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Bx41 fluorescence microscope, by Olympus (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti cd31, by Santa Cruz Biotechnology (1 mentions)
Anti claudin 5, by Abcam (1 mentions)
Ix71 digital camera, by Olympus (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Anti mbp, by Abcam (1 mentions)
Anti ctbp2, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti smi32
1
Immunohistochemical Staining of Brainstem
2
Immunostaining and Mitochondrial Imaging in Fibroblasts and Motor Neurons
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Fibroblasts or MNs were fixed in 3.7% formaldehyde at 37 C for 15 minutes, permeabilized and blocked as previously described. Fibroblasts were incubated with primary antibody Anti-TOM20 (1:50, Sigma) overnight, and then, after washing, with antirabbit Alexa Flour 594 (1:1000, Life Technologies) secondary antibody for 1.5 h at room temperature. The nuclei were stained with 200 nM DAPI (Sigma). MNs were also incubated with primary antibody Anti-SMI32 (1.100, Covance) overnight, and then, after washing, with anti-mouse Alexa Flour 488 (1:1000, Life Technologies) secondary antibody for 1.5 h at room temperature. The TOM20 fluorescence intensity and distribution expression were quantified using ImageJ software (http://rsbweb.nih. gov/ij/; date last accessed July 17, 2016) (79, 80) . These data were calculated in 10 randomly selected fields/well (3 wells/condition/experiment in four to five experiments). For mitochondrial and lysosome staining in MNs, the cells were infected with CellLightV R Mitochondria-RFP BacMam 2.0 (Life Technologies) and CellLightV R lysosome-GFP BacMam 2.0 (Life Technologies). After 48 h, the cells were fixed with 4% formaldehyde solution in PBS for 20 minutes at room temperature and stained with DAPI (200 nM, Sigma).
3
Histological Analysis of Immune Markers
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Reagents were purchased from Sigma (Italy). Antibodies raised in rabbit: anti‐arginase 1 (ARG1) (1:100, Abcam, UK); anti‐CD163 (1:100, Santa Cruz Biotechnology, USA); anti‐choline acetyltransferase (1:500, AbCam); anti‐DAO1 (1:200, Bioss, USA); anti‐H1R (1:200, Alomone, Israel); anti‐H2R (1:200, MyBioSource, USA); anti‐H3R (1:200, Alomone); anti‐H4R (1:200, Santa Cruz Biotechnology); anti‐HDC (1:20, AbCam); anti‐HNMT (1:200, Sigma); anti‐inducible nitric oxide synthase (iNOS) (1:1000, CST, USA); anti‐myelin basic protein (MBP) (1:1000 CST); anti‐nuclear factor‐kappa B (NF‐κB) p65 (1:500, CST); anti‐phospho‐AKT (1:500, CST); anti‐phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, CST); and anti‐phospho‐NF‐κB p65 (Ser536) (1:500, CST). Antibodies raised in mouse: anti‐AKT (1:500, Santa Cruz Biotechnology); anti‐GAPDH (1:2500, Calbiochem, USA); anti‐gp91phox (1:1000, BD Transduction Laboratories, USA); anti‐SMI32 (1:1000, Covance, USA); and anti‐p44/42 MAPK (ERK1/2) (L34F12) (1:1000, CST). Antibodies raised in rat: anti‐CD11b (1:200, AbD Serotec, UK).
4
Immunocytochemistry for Motor Neuron Survival
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Immunocytochemistry was run as previously described [35 (link), 39 (link)]. After blocking the non-specific binding sites by incubation with a solution containing 10% normal goat serum (NGS) and Triton in PBS (phosphate-buffered saline) 0.01 M, the cells were incubated with the primary antibodies (overnight at 4 °C), diluted in a solution containing 1% NGS and Triton in PBS 0.01 M. We used the following primary antibodies: anti-SMI32 (1:1000, Covance, Princeton, NJ, USA) and anti-NeuN (1:250, Chemicon, Temecula, CA, USA). Cells were then washed and incubated with the appropriate secondary fluorescent antibody (1:500, Alexa Fluor Dyes, Life Technologies, Grand Island, NY, USA) or secondary biotinylated antibody (1:500, Vector Laboratories, Burlingame, CA, USA) for tyramide signal amplification (TSA, Perkin Elmer) following the manufacturer’s instructions. Images were acquired with Olympus BX41 fluorescence microscope. Motor neuron survival was evaluated as previously described [35 (link), 39 (link)]. The labeling with anti-SMI32 antibody highlights motor neurons with typical morphology and large cell bodies (diameter ≥ 20 μm) and anti-NeuN antibody was used to identify all neurons in twelve adjacent frames per well at × 10 magnification. Data were expressed as the ratio of the number of motor neurons to the total neurons.
5
Immunohistochemical Analysis of Mouse Brain
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The mice were sacrificed by cardiac perfusion with 0.9% saline, and their brains were cut into 20 μm coronal sections after being fixed in 4% paraformaldehyde. The brain sections were incubated with anti‐MBP (1:400, Abcam), anti‐SMI32 (1:400, Covance), anti‐CD31 (1:200, Santacruz), anti‐claudin‐5 (1:400, Abcam), anti‐occludin (1:400, Invitrogen), anti‐GFAP (1:400, CST), anti‐MPO (1:400, CST), anti‐MMP‐9 (1:400, CST) antibodies, respectively, overnight at 4°C. After being washed with PBS, the sections were incubated with the appropriate secondary antibodies for 2 h in the dark at room temperature. The sections were imaged using an Olympus microscope with an IX71 digital camera (Olympus), and the images were quantified with IPP 6.0 software. All evaluations were performed by a blinded investigator.
6
Immunohistochemical Analysis of Retinal Tissues
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The immunohistochemical methods have been described previously.28 (link),31 (link),32 (link) Eyecups were fixed with 4% paraformaldehyde, cryoprotected, and embedded in tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA, USA), and 10- to 15-µm-thick frozen sections were cut. The tissues were incubated with diluted primary antibodies overnight (sections) or for 48 hours (whole mounts), washed, and then incubated in secondary antibodies for 2 to 4 hours. Images of immunolabeled retinal sections or whole mounts from control and experimental groups were obtained with an Olympus FV1200 MPE confocal microscope (Olympus, Tokyo, Japan) using a 40× oil-immersion objective. High-resolution (1024 × 1024 pixels) z-stack images were taken using step sizes of 0.7 to 2.0 µm and compiled. The brightness and contrast of micrographs were adjusted using Photoshop CS6 (Adobe, San Jose, CA, USA). The primary antibodies were anti-PKCα (1:10,000; Sigma-Aldrich); anti-Chx10 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA); anti-calbindin (1:500; Santa Cruz Biotechnology); anti-SMI32 (1:2000; Covance, Princeton, NJ, USA); anti-VGlut1 (1:150; BioLegend, San Diego, CA, USA); and anti-CtBP2 (1:500, Santa Cruz Biotechnology). Secondary antibodies were conjugated with Alexa Fluor 488, 594, and 633 (1:200; Thermo Fisher Scientific, Waltham, MA, USA).
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