On January 5, 2017Mohammad Baragji, University of Copenhagen wrote:
5
Single fluorescent staining targeting Parvalbumine expressing interneurons.
Primary antibody: Mouse Mab Clone 58E1, OYSTER-488-LABELED [Synaptic Systems, cat: 195 011C2], 1:200 and 1:600 dilution.
Protocol in short:
Sections were washed (3x10min), treated with target retrieval solution and washed again (1×10 min) Sections were then rinsed in FAB blocking buffer with Donkey anti-mouse followed by incubation with a fluorescent 488-labeled antibody directed towards PV (SYNAPTIC SYSTEMS) for 5 days. Sections were then washed (3×10 min) and (1×10 min) in DAPI and mounted on slides, air-dried and then mounted with cover slips.
Results:
Staining appears specific, as PV staining is intense around cell nuclei stained with DAPI. The PV antibody is thus appropriate for staining PV interneurons. The staining results further shows that both the 1:200 and 1:600 concentration are suitable.
Western blot
Immunostaining
Not Rated
IP
Not Rated
ELISA
Not Rated
Flow cytometry
Not Rated
Luminex
Not Rated
On January 5, 2017Mohammad Baragji, University of Copenhagen wrote:
Protocol in short:
In short, sections were washed (3x10min), treated with target retrieval solution and washed again (1×10 min). Sections were then rinsed in FAB blocking buffer with Donkey anti-mouse followed by incubation with a rabbit antibody directed towards GABA for 2 days. Sections were then washed (3×10 min) and incubated for 4 hours with a secondary antibody, Donkey-anti-Rabbit with a fluorescent 568-labeled directed towards the primary GABA antibody. The second primary antibody 488-labeled directed towards PV was added and sections were incubated for 5 days. Sections were then washed (4×15 min) and (1×10 min) in DAPI. Finally, the sections were mounted on slides, air-dried and then mounted with cover slips.
Results:
The double staining results show that the double staining with both antibodies at the same time was not successful. The single stainings appear to be specific, but when both antibodies are combined, the GABA antibody loses its specificity.
Western blot
Not Rated
Immunostaining
IP
Not Rated
ELISA
Not Rated
Flow cytometry
Not Rated
Luminex
Not Rated
On October 3, 2014Simon Mølgaard, Mayo Clinic wrote:
5
This antibody is primary labelled to oyster 488. It has been used on cryo-sections with a thickness up to 150 um with great succes. This antibody shows high specificity with very little background.
On January 5, 2017 Mohammad Baragji, University of Copenhagen wrote:
Single fluorescent staining targeting Parvalbumine expressing interneurons.
Primary antibody: Mouse Mab Clone 58E1, OYSTER-488-LABELED [Synaptic Systems, cat: 195 011C2], 1:200 and 1:600 dilution.
Protocol in short:
Sections were washed (3x10min), treated with target retrieval solution and washed again (1×10 min) Sections were then rinsed in FAB blocking buffer with Donkey anti-mouse followed by incubation with a fluorescent 488-labeled antibody directed towards PV (SYNAPTIC SYSTEMS) for 5 days. Sections were then washed (3×10 min) and (1×10 min) in DAPI and mounted on slides, air-dried and then mounted with cover slips.
Results:
Staining appears specific, as PV staining is intense around cell nuclei stained with DAPI. The PV antibody is thus appropriate for staining PV interneurons. The staining results further shows that both the 1:200 and 1:600 concentration are suitable.
On January 5, 2017 Mohammad Baragji, University of Copenhagen wrote:
Double staining targeting Parvalbumine expressing interneurons.
Primary antibody: Mouse Mab Clone 58E1, OYSTER-488-LABELED [Synaptic Systems, cat: 195 011C2]
Secondary antibody: Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 568 conjugate [Life technologies Cat: A10042]
Protocol in short:
In short, sections were washed (3x10min), treated with target retrieval solution and washed again (1×10 min). Sections were then rinsed in FAB blocking buffer with Donkey anti-mouse followed by incubation with a rabbit antibody directed towards GABA for 2 days. Sections were then washed (3×10 min) and incubated for 4 hours with a secondary antibody, Donkey-anti-Rabbit with a fluorescent 568-labeled directed towards the primary GABA antibody. The second primary antibody 488-labeled directed towards PV was added and sections were incubated for 5 days. Sections were then washed (4×15 min) and (1×10 min) in DAPI. Finally, the sections were mounted on slides, air-dried and then mounted with cover slips.
Results:
The double staining results show that the double staining with both antibodies at the same time was not successful. The single stainings appear to be specific, but when both antibodies are combined, the GABA antibody loses its specificity.
On October 3, 2014 Simon Mølgaard, Mayo Clinic wrote:
This antibody is primary labelled to oyster 488. It has been used on cryo-sections with a thickness up to 150 um with great succes. This antibody shows high specificity with very little background.
I used this at a concentration of 1:200.