Immunocytochemistry/ Immunofluorescence abreview for Anti-GFAP antibody [GF5] - Astrocyte Marker

Excellent
Abreviews
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Stimulated Cor1 cells)
Specification
Stimulated Cor1 cells
Fixative
Formaldehyde
Permeabilization
Yes - TBS/BSA/azide/0.3% Triton X
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C

Other product details

Dilution
1/1500
Incubation time
2 hour(s) and 0 minute(s) · Diluent: TBS/BSA/azide/0.3% Triton

Secondary antibody

Name
Non-Abcam antibody was used: anti mouse IgG
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Dilution
1/1000

Additional data

Additional Notes
I co- incubated stimulated Cor1 cells with Abcam's and a competitor anti-GFAP Abs: after washing in TBS x3 for 15minutes I then incubated the cells with buffer containing Goat anti Rabbit -Alexa 594, Goat anti Mouse Alexa- 488 and Hoechst for 1 hr at RT diluted in same buffer solution as the primary Abs.
After further TBS washes, coverslips were picked up,rinses in distilled water performed to get rid of buffer salts and then the coverslips were mounted on glass slides, using a homemade "anti-fade" hard-setting mountant.
Images were captured using a Zeiss Apotome Microscope system: Z-stacks of each channel were flattened in "cut-view".
Adobe's Photoshop was used to merge red/greenchannels with Hoechst and then to globally enhance the composite image, using "unsharp mask".

Seems to me that both Abs are co-localised. I did this because, in FFPW sections, the positivity seen with these Abs in the svz of mouse/rat brain FFPW sections of lateral ventricle is not identical (in my hands).
My point therefore, is that one must always test several Abs raised against any particular protein unless Peer review concludes that one particular Ab reagent is agreed to be specific for any cell/tissue preparation.

The cells were seeded/grown on coverslips and then fixed by Philipp Suetterlin, PhD student Wolfson Centre for Age-Related Diseases, Kings College London ...thanks, Phil! All previous Abreviews using Cor1 cells were possible only because of Phil's excellent skills in growing/maintaining/fixing Cor1 cells.

Mr. Carl Hobbs

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Submitted Apr 15 2009

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