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An optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue
- Source :
- MethodsX, Vol 5, Iss, Pp 1-7 (2018), MethodsX
- Publication Year :
- 2018
- Publisher :
- Elsevier BV, 2018.
-
Abstract
- Graphical abstract<br />Netrin-1, an axon guidance protein, is difficult to detect using immunohistochemistry. We performed a multi-step, blinded, and controlled protocol optimization procedure to establish an efficient and effective fluorescent immunohistochemistry protocol for characterizing Netrin-1 expression. Coronal mouse brain sections were used to test numerous antigen retrieval methods and combinations thereof in order to optimize the stain quality of a commercially available Netrin-1 antibody. Stain quality was evaluated by experienced neuroanatomists for two criteria: signal intensity and signal-to-noise ratio. After five rounds of testing protocol variants, we established a modified immunohistochemistry protocol that produced a Netrin-1 signal with good signal intensity and a high signal-to-noise ratio. The key protocol modifications are as follows: • Use phosphate buffer (PB) as the blocking solution solvent. • Use 1% sodium dodecyl sulfate (SDS) treatment for antigen retrieval. The original protocol was optimized for use with the Netrin-1 antibody produced by Novus Biologicals. However, we subsequently further modified the protocol to work with the antibody produced by Abcam. The Abcam protocol uses PBS as the blocking solution solvent and adds a citrate buffer antigen retrieval step.
- Subjects :
- Signal intensity
0301 basic medicine
Immunofluorescence
Clinical Biochemistry
PB, phosphate buffer
Phosphate buffer
AF, Alexa Fluor
chemistry.chemical_compound
0302 clinical medicine
NDS, normal donkey serum
Netrin
TBS, tris-buffered saline
Sodium dodecyl sulfate
Bovine serum albumin
lcsh:Science
medicine.diagnostic_test
biology
HRP, horseradish peroxidase
Brain
SNR, signal-to-noise ratio
3. Good health
Medical Laboratory Technology
Antigen retrieval
RT, room temperature
Immunohistochemistry
IHC, immunohistochemistry
SDS, sodium dodecyl sulfate
PBS, phosphate-buffered saline
Stain
03 medical and health sciences
medicine
IF, immunofluorescence
Citrate buffer
ComputingMethodologies_COMPUTERGRAPHICS
Protocol (science)
030102 biochemistry & molecular biology
TB, tris buffer
chemistry
biology.protein
BSA, bovine serum albumin
Signal-to-noise ratio
lcsh:Q
DAPI, 4′, 6-diamidino-2-phenylindole
030217 neurology & neurosurgery
Neuroscience
TH, tyrosine Hydroxylase
Biomedical engineering
Subjects
Details
- ISSN :
- 22150161
- Volume :
- 5
- Database :
- OpenAIRE
- Journal :
- MethodsX
- Accession number :
- edsair.doi.dedup.....9c358f1e90c82f17c473a14934b2f90b