I preface my response by saying my immuno-TEM experience is only in mouse brain tissue.  I have not worked with blood or blood components.

What is your general protocol?  Which fixatives/buffers are you using?  What method do you use to permeabilize the plasma membrane? How long is your primary antibody incubation? Are you doing a second fixation step after the antibody labeling so that the primary and secondary AB are fixed together?  How long is your silver enhancement reaction?

Answering these questions will help pinpoint where the problem is.

Hi Travis,

Thank you very much for your response. I am using following protocol for immuno-tem. Following protocol is well defined and clearly written. But I don't know where I am going wrong. Just one change I have made in the attached protocol. I have used 4% PFA plus 0.1% - 0.2% glutaraldehyde during the first fixation.   

Durgesh,

Here are some things you can try to improve your results: Omit the glutaraldehyde from your primary fixative as it may be damaging to your antigen's epitope.  You can also try increasing your antibody incubation times.  I did an overnight incubation at 4C for thick sections of tissue.  That extreme is not necessary for your cell suspensions, but maybe a couple of hours would be beneficial.  Your protocol states that membrane permeabilization is not necessary due to the 10 µm thickness of the cryosections, but perhaps for your particular antibody, a membrane perm. step would be helpful to facilitate antibody penetration.  This is not the ideal condition for preserving ultrastructure, however.  If none of those suggestions are helpful, it may be necessary to try out some other primary antibodies.  The fact that an antibody works for confocal is not a guarantee that it will be successful in TEM.  

Hope these ideas are helpful to you. I'd like to hear back about how you ultimately solve the problem.