three color staining - (Feb/06/2007 )
Does anyone have experience with three color staining. I am considering doing a triple labeling for F-actin, microtubule and nuclei but don't have a protocol on hand. If you have done that before, could you kindly post one? Thanks a lot!
esmile
-esmile-
QUOTE (esmile @ Feb 7 2007, 03:37 AM)
Does anyone have experience with three color staining. I am considering doing a triple labeling for F-actin, microtubule and nuclei but don't have a protocol on hand. If you have done that before, could you kindly post one? Thanks a lot!
esmile
esmile
it depends, among others, on how your are equipped with different LASREs or filters, respectively; for F-actin we use Alexafluor 633-phalloidin, for nucleus DAPI (UV immission), and for microtubuli an Ab with secondary labeling by Alexa-fluor 488; one could also take Alexa fluor 546 for a fourth labelling
-The Bearer-
QUOTE (The Bearer @ Feb 7 2007, 02:20 AM)
QUOTE (esmile @ Feb 7 2007, 03:37 AM)
Does anyone have experience with three color staining. I am considering doing a triple labeling for F-actin, microtubule and nuclei but don't have a protocol on hand. If you have done that before, could you kindly post one? Thanks a lot!
esmile
esmile
it depends, among others, on how your are equipped with different LASREs or filters, respectively; for F-actin we use Alexafluor 633-phalloidin, for nucleus DAPI (UV immission), and for microtubuli an Ab with secondary labeling by Alexa-fluor 488; one could also take Alexa fluor 546 for a fourth labelling
Thank you for the information. When you fix the cells, what kind of fixative do you use? I have read that methanol might disrupt actin filament while formaldehyde is not good for preserving the microtubule structure.
-esmile-
QUOTE (esmile @ Feb 16 2007, 09:51 PM)
QUOTE (The Bearer @ Feb 7 2007, 02:20 AM)
QUOTE (esmile @ Feb 7 2007, 03:37 AM)
Does anyone have experience with three color staining. I am considering doing a triple labeling for F-actin, microtubule and nuclei but don't have a protocol on hand. If you have done that before, could you kindly post one? Thanks a lot!
esmile
esmile
it depends, among others, on how your are equipped with different LASREs or filters, respectively; for F-actin we use Alexafluor 633-phalloidin, for nucleus DAPI (UV immission), and for microtubuli an Ab with secondary labeling by Alexa-fluor 488; one could also take Alexa fluor 546 for a fourth labelling
Thank you for the information. When you fix the cells, what kind of fixative do you use? I have read that methanol might disrupt actin filament while formaldehyde is not good for preserving the microtubule structure.
we use 4% formaldehyde and 4% sucrose in PBS to fix and get nice results for microtubuli,
there might be differences for specific cell lines, so you better optimize a protocol
-The Bearer-