Application
Immunohistochemistry free floating
Sample
Mouse Tissue sections (Mouse brain and spinal cord)
Specification
Mouse brain and spinal cord
Other product details
Incubation time
48 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: PBS-T with 2% NGS
Dilution
1/2000
Secondary antibody
Dilution
1/500
Name
Non-Abcam antibody was used: Jackson AffiniPure Donkey Anti-Sheep IgG secondary
Host species: Donkey
Clonality: Polyclonal
Conjugation: Biotin
Host species: Donkey
Clonality: Polyclonal
Conjugation: Biotin
Additional data
Additional Notes
Our mouse brain and spinal cord samples were fixed in PFA (2h30 fixation time) and stored in a 30% sucrose solution. A freezing microtome was used to cut 30 ´m sections, which were used for free-floating immunohistochemistry.
Details:
Washing solutions: PBS 0,1M and PBS-T
Peroxidase inhibition: 15 minutes with H2O2 1% in PBS
Blocking non-specific binding: 2 hours in 30mg glycine + 3,6mL PBS-T + 400´L NGS
Primary antibody: Abcam ab19353 diluted in PBS-T with 2% NHS and tested concentrations of 1/2000, 1/8000.
Secondary antibody: Jackson AffiniPure Donkey Anti-Sheep IgG diluted 1/500, incubated for 1 hour at room temperature
ABC incubation: 1/200 reagent A and 1/200 reagent B in PBS-T
Revelation with DAB for over 30 minutes with no results
Ending DAB reaction with Tris HCl 0,05M pH 7,6.
In the images provided, we show the concentrations 1/8000 (brain), 1/2000 (spinal cord) and one of our controls with tyrosine hydroxylase 1/6000 in mouse brain. The Th antibody has shown us good results using our protocol for free-floating. However, the Dbh never did under the same conditions.
(Also tested the concentration 1/100, 1/500, 1/2000 for immunofluorescence with no results)
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Abcam user community
Verified customer
提交于 Feb 22 2017