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Author Topic: Troubleshooting ideas for selective staining  (Read 4909 times)

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Offline yasiangel

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Troubleshooting ideas for selective staining
« on: April 04, 2007, 04:28:52 PM »
We are trying to figure out how to make our primary antibody, HuNu (chemicon MAB 1281), work properly.   The antibody stains for the human nucleus, however, the staining is selective and not all of the nuclei are stained.  Our goal is to identify human stem cells transplanted into rat tissue.  When we noticed that the staining was very faint in some cells, we moved to a more simplified system in order to optimize the staining procedure.  Human stem cells were embedded within a 3-D agarose gel system, fixed in 4% para for an hour, then cryosectioned.  As you can see from our pictures, the HuNu staining is selective and inconsistent. 

A description of our protocol is given below.
 
Primary Antibodies:
HuNu (chemicon MAB 1281)
 
Seconday Antibody:
Fluorescent labeled secondary Ab used is Alexa-flouro 546 (red) (starting concentration 2 mg /ul)

This is the protocol that we used:

1. Rinse slides in PBS to rehydrate 3 times @ 5 min each
2. blocking with 8% goat serum & 0.4% Triton X solution for 1 hour
3. Rinse with 1 quick PBS wash and PBS tapped off and air dried to remove excess moisture
4. Specimens circled with PAP pen and primary Ab added
5. Refrigerated overnight
6. Afterwards, excess solution tapped off and washed with PBS 3 times @ 5 min each
7. Secondary Ab centrifuged, diluted, and added to slides (flourescence)
 
Hoechst solution 1:2500
goat anti-mouse secondary Ab 1:500
2% Goat serum


8. Incubate in humidity chamber @ room temp 2 hours
9. Rinse once quickly in PBS, then rinse again 3 times @ 5 min each
10.  Place cover slip and store overnight in dark
 
We have tried a dilution series of the primary antibody
1:100
1:50
1:25
 
And also a dilution series for the secondary antibody
1:500
1:250
1:100
 
In all of the cases above, the staining was extremely light.  The best combination of primary dilution & secondary dilution was:
 
Primary AB Dilution 1:25
Secondary AB Dilution 1:100
 
As seen below:
 
 
Hoechst counter stain


 
Fluorescent labeled secondary AB
 
 

Co-localized secondary AB w/ Hoechst


 
 
Does anyone have suggestions for improving the quality of the staining?  For example, Should we incubate the primary antibody longer? If so how long?

Alternatively, does anyone know of a more consistent primary antibody we can use to identify human cells in rat tissue?

Thank you!


Troubleshooting ideas for selective staining
« on: April 04, 2007, 04:28:52 PM »

Offline vega07

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Re: Troubleshooting ideas for selective staining
« Reply #1 on: April 04, 2007, 05:32:21 PM »
I would try these things. If you really need to do immunofluorecense, in order to increase your signal you can try a biotinylated-antibody followed by a streptavidin-conjugated fluorochrome. But since the level of your antigen looks low, why don't you try IHC. I think that it would be more reliable.

Good luck.

Offline Crispinator

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Re: Troubleshooting ideas for selective staining
« Reply #2 on: September 09, 2010, 12:52:16 PM »
Did you ever figure out a solution to this? We are having the same problems  :-\

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Re: Troubleshooting ideas for selective staining
« Reply #2 on: September 09, 2010, 12:52:16 PM »