Anti-Tyrosine Hydroxylase 抗体 - Neuronal Marker (ab41528)

Anti-Tyrosine Hydroxylase antibody - Neuronal Marker 画像

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  Western blot - Anti-Tyrosine Hydroxylase antibody - Neuronal Marker (ab41528)
  All lanes : Anti-Tyrosine Hydroxylase antibody - Neuronal Marker (ab41528) at 1 µg/ml
  
  Lane 1 : Mouse Substantia Nigra at 5 µg
  Lane 2 : Rat Substantia Nigra at 10 µg
  Lane 3 : Mouse Dopaminergic Nucleus at 10 µg
  Lane 4 : Rat Dopaminergic Nucleus at 10 µg
  
  Secondary
  Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
  Developed using the ECL technique
  
  Performed under reducing conditions.
  
  Predicted band size : 59 kDa
  Observed band size : 60 kDa (why is the actual band size different from the predicted?)
  Additional bands at : 70 kDa. We are unsure as to the identity of these extra bands.
  
  Exposure time : 30 seconds
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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tyrosine Hydroxylase antibody - Neuronal Marker (ab41528)

  IHC image of Tyrosine Hydroxylase staining in Rat 6 week brain (coronal) formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab41528, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

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  Immunocytochemistry/ Immunofluorescence - Tyrosine Hydroxylase antibody (ab41528)

  ICC/IF image of ab41528 stained rat PC12 cells. The cells were methanol fixed (5 min), permabilised in PBS-T (20 min) and incubated with the antibody (ab41528, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

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  Immunohistochemistry (PFA perfusion fixed frozen sections) - Tyrosine Hydroxylase antibody - Neuronal Marker (ab41528)Image from Ettrup KS et al, J Chem Neuroanat. 2010 May;39(3):151-65. Epub 2009 Dec 28, Fig 2, doi:10.1016/j.jchemneu.2009.12.004.
  Minipigs were deeply anesthetized with a combination of midazolam and ketamine, prior to transcardial perfusion with phosphate buffered 4% paraformaldehyde (pH 7.4). After perfusion, the brains were removed with special care taken to preserve the optic chiasm and the median eminence. Blocks of tissue containing the hypothalami were dissected, postfixed in the same fixative for 1 day and subsequently cryoprotected in 30% sucrose for 3–4 days, prior to freezing. 10 series of 40-mm thick coronal (6 animals), sagittal (1 animal), and horizontal (1 animal) cryostat sections were collected. Coronal sections for immunohistochemistry were maintained at -18°C as free-floating sections in a cryoprotectant poly-ethylene glycol solution for up to four weeks.
  Immunohistochemistry was performed using the avidin-biotin method. Accordingly, free-floating sections were first rinsed in Tris-buffered saline (TBS; 0.05 M; pH 7.4) for 15 minutes. Incubations with avidin (0.1%) and biotin (0.01%) were each of 10 minute duration and each procedure were followed by a 2 minute TBS-rinse. The sections were preincubated with 1% Triton X- 100 and 0.2% milk in TBS for 30 minutes, prior to incubation with the primary antibody, ab41528, for 72 hours at 4°C at a 1/400 dilution. The sections were then washed three times 15 minutes in TBS and 1% Triton X-100 prior to incubation for 1 hour at room temperature with the secondary anti-rabbit biotinylated antibody at a 1/400 with TBS that contained 1% Triton X- 100 and 0.2% milk. After a brief rinse in TBS, the endogenous peroxidase activity was blocked with a solution of 10 ml hydrogen peroxide, 10 ml methanol, and 80 ml TBS, for 10 minutes. The sections were then rinsed in TBS and 1% Triton for three times 15 minutes and incubated for 1 hour at room temperature with avidin-peroxidas diluted 1/400 in TBS that contained 1% Triton X-100 and 0.2% milk. The sections were then rinsed three times for 15 minutes in TBS and 1% Triton, prior to avidin-peroxidase visualization. The latter step consisted of incubation for 10 minutes in 10ml water in which one diaminobenzidine tablet had been dissolved and to which 10 ml of 35% H₂O₂ had been added. Finally, the sections were dehydrated, mounted and coverslipped with Depex.
  
  PHA: posterior hypothalamic area.
  
  VMH: ventromedial hypothalamic nucleus.
  
  Arc: arcuate nucleus.