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Immunofluorescence - unspecific staining - (Feb/20/2012 )

Hi,

I stained some cells on cover slips (immunofluorescence) and got quite unspecific staining for my antibody, which is rather surprising since the same antibody has worked on immunofluorescence slides with other cells before.
Here's the outline of my protocol:

Fixation was done with 4% PFA for 20 min
Permeabilization with 0,1% Tx-100 for 4 min
Blocking with 5 % Normal Goat Serum in PBS for 1h at RT.
Primary AB incubation o/n at 4C, 1:100
Secondary AB for 1h at RT, 1:200
DAPI 1:1000 (DAPI staining worked well)
Between the steps respectively 3x washing with 0,05 % Tween/PBS

Have you got any suggestions or ideas how I could improve the staining ?
Thanks a lot :)

-Tabaluga-

Tabaluga on Mon Feb 20 12:00:01 2012 said:


Hi,

I stained some cells on cover slips (immunofluorescence) and got quite unspecific staining for my antibody, which is rather surprising since the same antibody has worked on immunofluorescence slides with other cells before.
Here's the outline of my protocol:

Fixation was done with 4% PFA for 20 min
Permeabilization with 0,1% Tx-100 for 4 min
Blocking with 5 % Normal Goat Serum in PBS for 1h at RT.
Primary AB incubation o/n at 4°C, 1:100
Secondary AB for 1h at RT, 1:200
DAPI 1:1000 (DAPI staining worked well)
Between the steps respectively 3x washing with 0,05 % Tween/PBS

Have you got any suggestions or ideas how I could improve the staining ?
Thanks a lot

perhaps if you titrate both the primary and secondary antibodies, you'd find the optimal concentration thereby reducing the background ((esp the secondary- 1:200 is a bit much imo even for immunocyt). I suppose you also ran the appropriate controls eg only secondary Ab...

-casandra-

casandra on Mon Feb 20 19:52:54 2012 said:


perhaps if you titrate both the primary and secondary antibodies, you'd find the optimal concentration thereby reducing the background ((esp the secondary- 1:200 is a bit much imo even for immunocyt). I suppose you also ran the appropriate controls eg only secondary Ab...


Thanks. I did not have enough material for performing controls, but I will certainly include them in the next run. As for the concentration, we've always been taking 1:200 for the secondary AB and with the other cells, it worked perfectly. Perhaps I should indeed take the time and titrate though...

-Tabaluga-

Was the background green by any chance? If so, you need to wash the coverslips more to remove residual free formaldehyde before proceeding with the staining procedure.

As you block in NGS, I presume the antibody is a goat primary?

-bob1-

bob1 on Wed Feb 22 22:18:47 2012 said:


As you block in NGS, I presume the antibody is a goat primary?

we use ngs as block when we use a goat secondary.

-mdfenko-

@bob1: No, the background was not generally green, but rather the color of the respective AB. (I had three primary AB's, but on different cover slips except for the one where I costained.) I know formaldehyde can give background, so I took great care with the washing.

@mdfenko: My secondary ABs were indeed goat (and one of them was donkey, I think - not sure right now). Primary ABs were mouse, rat and rabbit.

-Tabaluga-

Tabaluga on Wed Feb 22 19:39:05 2012 said:


casandra on Mon Feb 20 19:52:54 2012 said:


perhaps if you titrate both the primary and secondary antibodies, you'd find the optimal concentration thereby reducing the background ((esp the secondary- 1:200 is a bit much imo even for immunocyt). I suppose you also ran the appropriate controls eg only secondary Ab...


Thanks. I did not have enough material for performing controls, but I will certainly include them in the next run. As for the concentration, we've always been taking 1:200 for the secondary AB and with the other cells, it worked perfectly. Perhaps I should indeed take the time and titrate though...

Have you tried incubating the primary Ab just for one or two hours at RT? I know..I know...your protocol worked perfectly with the other cells but it's worth a try anyhow...:)

-casandra-

I think what I need is just to perform a few test runs (vary AB concentration, incubation etc.) to see what will work out best. And of course, controls. Problem is just that till my cells are "ready", it takes quite some time (due to the experimental setup which this IF staining is actually part of). Anyway, I'll take the time.

Thanks for your replies =)

-Tabaluga-

mdfenko on Thu Feb 23 20:09:31 2012 said:


bob1 on Wed Feb 22 22:18:47 2012 said:


As you block in NGS, I presume the antibody is a goat primary?

we use ngs as block when we use a goat secondary.

I didn't know. I always used BSA

-Curtis-

Curtis on Mon Feb 27 17:27:18 2012 said:


mdfenko on Thu Feb 23 20:09:31 2012 said:


bob1 on Wed Feb 22 22:18:47 2012 said:


As you block in NGS, I presume the antibody is a goat primary?

we use ngs as block when we use a goat secondary.

I didn't know. I always used BSA

we block with a mixture of ngs and bsa and use ngs in our antibody solutions when we use a secondary raised in goat (that way, any species of antibody that would react with goat proteins will be preabsorbed).

-mdfenko-