Protocol Online logo
Top : Forum Archives: : Cell Biology

Fibroblast staining - (Oct/28/2006 )

Hi

Is there a specific protocol for Fibroblast staining. I have isolated lung fibroblasts from wt and ko mice and tried staining them according to the staining protocol i used. But my antibody did not work. IS there a specific protocol for staining.

Thanks! smile.gif

-pmenon-

QUOTE (pmenon @ Oct 29 2006, 01:59 AM)
Hi

Is there a specific protocol for Fibroblast staining. I have isolated lung fibroblasts from wt and ko mice and tried staining them according to the staining protocol i used. But my antibody did not work. IS there a specific protocol for staining.

Thanks! smile.gif


from your question it is not quite clear what you really try to stain; if you are in search for a fibroblast-specific reference protein, try FSP1 (fibroblast specific protein 1); it is used in IHC to distinguish fibroblast form other cell types;


quotation:
STRUTZ F, OKADA H, LO CW, et al: Identification and characterization
of a fibroblast marker: FSP1. J Cell Biol 130:393–405, 1995

-The Bearer-

Sorry, if i was not clear.
I had tried the staining protocol i used for smooth muscle cells in which my antibody seem to work fine. But when tried the same protocol for the wt and ko fibroblast my antibody staining did not work.
That was what made me wonder if there is a specific staining protocol for fibroblast cells.

This is the protocol
3.7% formaldehyde fixing- 10 min
0.2% Triton-PBS- 10 min
Blocking : 1% BSA, 5% goat serum in 1X PBS - 30 min at RT
1 Ab (rabbit poly): 1: 100( 3 hrs at RT)
2 Ab ( goat anti rabbit, molecular probes) 1: 3000 ( 1 hr @ RT)
Store in PBS o/N

Additioanlly this time i stained for Rhodamine Philloidin( 2hrs) and DAPI ( 10 min) the next day before i took the images.
Could philloidin staining have affected my antibody?( This is the first time in have tried out philloidin staining after staining for my protein)

Thanks in advance for the reply!
smile.gif










QUOTE (kosmodrom @ Oct 29 2006, 04:54 AM)
QUOTE (pmenon @ Oct 29 2006, 01:59 AM)

Hi

Is there a specific protocol for Fibroblast staining. I have isolated lung fibroblasts from wt and ko mice and tried staining them according to the staining protocol i used. But my antibody did not work. IS there a specific protocol for staining.

Thanks! smile.gif


from your question it is not quite clear what you really try to stain; if you are in search for a fibroblast-specific reference protein, try FSP1 (fibroblast specific protein 1); it is used in IHC to distinguish fibroblast form other cell types;


quotation:
STRUTZ F, OKADA H, LO CW, et al: Identification and characterization
of a fibroblast marker: FSP1. J Cell Biol 130:393–405, 1995

-pmenon-