I think you could have fixing problems with the yeast wall. Your antibody does not bind efficiently cell surface, or your antigen is masked and the cells need first undergo some procedure before V5 conjugated FITC antibody  incubation. Search in pub-med some papers of immunofluorescence in yeast. You should also check that the microscope filter is suitable for the FITC emission.   Good luck

thanks Paola for your suggestion....I am using direct immunofluorescence (primary and secondary conjugated) protocol from invitrogen. Do you think I should switch to indirect staining method. 

Indirect staining method is more sensitive than direct, I think your problem is also the cell wall of yeast , look at some papers on pubmed

http://www.biochem.emory.edu/labs/acorbe2/immuno.html

Not all commercial antibodies work as expected even if they are sold for the intended application.  In addition, direct conjugation of flourophores to an antibody can affect its binding.  Try secondary staining but it you have to use direct conjugate for multiplexing or other reasons, try 2-3 different clones from other sources. 

Please read the protocol in the current protocols journal.

Cell Biology > Molecular Trafficking

Fluorescent Labeling of Yeast

J.J. Baggett1, J.D. Shaw1, C.J. Sciambi1, H.A. Watson1, B. Wendland1

1 Johns Hopkins University, Baltimore, Maryland

Publication Name:  Current Protocols in Cell Biology

Unit Number:  Unit 4.13

DOI:  10.1002/0471143030.cb0413s20

Online Posting Date:  November, 2003

GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Table of Contents

Materials

Literature Cited

Abstract

This unit describes the use of several different fluorescence methods for labeling yeast cells. It includes methods to label the vacuole, the actin cytoskeleton, and chitin deposits on cell walls (bud scars), as well as methods for visualizing specific proteins in live cells with GFP chimeras and in fixed cells by immunofluorescence.

Keywords: yeast; fluorescent labeling; vacuole; endocytic pathway; chitin

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online

Hope it helps.

Hi Ranu, thanks for your suggestion. The above said download is not available here, but only abstract is available. I appreciate if you could share the document.

Hello Kk:

Attached please find the protocol in PDF. Hope it helps. Ranu