| View previous topic :: View next topic |
| Author |
Message |
|
|
Post subject: Double staing: CK 18 / 5 / 7 respectively with oct 3/4
|
 |
|
I work with bovine material and my primary antibodies are oct 3/4 Sc-8629 (Goat ) and CK 18 ( Progene GP-CK18), CK 14 (Progene # GP-CK14) and CK 5 ( Progene # GP-CK5). All Antibodies worked well alone; GP stands for Ginea Pig. How can I double stain (for instance oct 3/4 and CK 1 The first step would be: choose an antibody against goat + FITC (mouse anti goat sounds good) and the second step is choosing a antibody against GP + cy5 (now this is more difficult; cannot use goat secondaries? Can I use donkey? My slides are cow tissue? How can I get around this problem??? We use AB's from Jackson ImmunoResearch for fluorescence).
Very grateful for your help! Margarete |
|
 Mon Feb 01, 2010 2:19 pm |
|
|
|
|
Post subject: Chicken secondaries?
|
|
|
would be my first suggestion but I am sure that at least hrn will have the most helpful suggestions.
At least to my simple mind, by using eg chicken anti GP green, anti Goat red one can do simultaneous primary and secondary incubations.
I will always do a double- labelling on one section and then single labelling on the next two sections, using the wrong secondary Ab, just to confirm that there is/is not any x-reactivity, anyway.
Why cy5.....unless for triple labelling when also using Hoechst/DAPI to stain nuclei?
Invitrogen's Alexa dyes are my recommendation: Alexa 488 and Alexa594 for good spectral separation.
I will be also interested to learn from others' suggestions.
carl |
|
| Mon Feb 01, 2010 5:49 pm |
|
|
|
|
Post subject:
|
|
|
Unfortunately I could not find chicken anti GP + Alexa Fluor dyes. They are all goat (impossible!) or rabbit, and this + FITC. For the second (goat) Ab I could also take rabbit anti goat + Alexa 594 or would this be too "strong" then? I am rather new in fluorescent dying  I do want to use my vectashield + DAPI. Thank you for support, Margarete |
|
| Mon Feb 01, 2010 9:53 pm |
|
|
|
|
Post subject:
|
|
|
| Another possibility, if you don't want green for your GP-bound target, would be to use a rabbit anti-GP secondary conjugated to AP (such as Invitrogen's 61-4622), with Vector Red as the substrate. Vector Red is fluorescent as well as visible in white light. |
|
| Wed Feb 03, 2010 12:28 am |
|
|
|
|
Post subject:
|
|
|
 Thank you for your kind help: I'll try the Rabbit anti GP + FITC and the Rabbit anti GT + Alexa594 - see what happens. Invitrogen said it (Alexa594) would not be stronger but last longer than FITC. That's ok as we use a confocal.
Margarete |
|
| Wed Feb 03, 2010 1:49 pm |
|
|
|
|
Post subject: Do not forget to...
|
|
|
Immunostain one section with Gp Ab and probe with Rb anti Gt and also immunostain the next section with Gt Ab and probe with rb anti GP.
?
That way you prove that they do not X-react?
Sure, they should not x-react but...prove it?
Also, using a Laser/Diode Confocal microscope does NOT mean that your signal will last longer...
Simply: you get better images cos you paid MUCH more money to increase the sensitivity of your system.
Maintaining signal strength depends upon many factors, imho.
Please disagree/inform me as I am not very familiar with LSM
Curious Carl. |
|
| Wed Feb 03, 2010 8:43 pm |
|
|
|
