hi,
the antibody i am using for staining mouse brain sections (cryosections) works wonderfully with DAB, however i am not getting any signal for fluorescence. my steps are same for both the stainings except for quenching and signal amplification steps are present for DAB staining but not in fluorescence. Is it that some antibodies which work for DAB might not work for IF?

please help

hi,
As far as I know, an antibody labeled to react with DAB does not have a fluorescent signal on it. I think you need a fluorescent tagged antibody such as FITC. I'm going to move this over to Antibody based technology category to see if you can get some more help.

Omai

sorry, should have mentioned earlier, i meant the primary antibody, for fluorescence i am using FITC labelled secondary from vector. tried at dilutions from 1:50 to 1:500 for primary and 1:100 to 1:1000 for secondary, still no signal

There are a few controls to do to rule out certain possibilities:

1. Did you do a control incubation with just the secondary antibody or no antibody at all and then do DAB? ie. Sometimes, certain tissues give a DAB positive stain without Abs. The best control would be an isotype control primary Ab then use your secondary plus DAB stain. If this is negative then the signals you see using your test primary Ab are real.

2. Fixing the sample: These are cryo sections, but did you fix before doing the immunofluorescence. I use methanol then acetone (both from the fridge, each for 5 mins). If the cells are not fixed then you can loose IF signal.

3. You mentioned that you did amplification steps for the DAB but not for IF. Could this be the reason?

Just a thought - Are your primary antibodies monoclonals made in mice or goat polyclonal antisera?

Gswetha,

The DAB method is the most sensitive IHC method and requires the least concentration of primary antibodies to detect the antigen. On the other hand, directly tagged secondary antibody is THE LEAST sensitive. The concentration of primary antibodies that works in DAB method usually way too low for use with the fluorescent-labelled secondaries and requires to be up to 100 fold higher to see the signal.

There is a great article by Gloria Hoffman published recently in Current Protocols of Neuroscience about the usage of primary antibodies for tissue staining and the methods for the amplification of the signal. There is a protocol for Immunofluorescence Detection using Fluorophore-Tagged Secondary Antibodies in this paper and various methods for amplification of the signal:

Hoffman GE, Le WW, Sita LV. The importance of titrating antibodies for immunocytochemical methods. Curr Protoc Neurosci. 2008 Oct;Chapter 2:Unit 2.12.

thanx a lot

will definitely check up the article