Shankhinur,

A few points:  Are you glow-discharging your grids to make them hydrophilic?  This will ensure adequate sample adsorption to the grid (older grids tend to by hydrophobic).  I would also do a quick hard fix (1% Ga cacodylate-filtered buffer, 5 min) after the secondary Aby rinse, then followed by another rinse, before stain.  I would not dry out the grid before negative staining (with UA make sure there is no phosphate left).  I normally stain with 1% uranyl acetate (filtered) 2x 1min drops, then blot the stain off from the side of the grid (and between your forceps).  The key is to leave a thin layer of UA to dry over the sample.

good luck,

Michael Delannoy

Shankhinur, Do you have any neg control? looking at the image, I don't see many background/nonspecific gold staining. To enhance the phage structure, I recommend Pre 2% UA staining after step 4 on your protocol. For the Neg stain, I would leave UA for at least 1 min. Good luck

Thank you Delannoy and Asghari. @ Delannoy- Before using the gold grid I usually give hydrophilic treatment but not fix by 1% Ga cacodylate-filtered buffer. I will try it next. 

@ Asghari- What should I use as negative control? I am novice in TEM experiment, so I have no idea. I followed Pre 2% UA staining after step 4 but no significant improvement. 

Seems that the Phage head does not pick up the staining. how about if you try pre-staining before step 2.

For Neg control; scape step 3 and 4 and see if there is any back ground gold staining. That will tell you whether you have any nonspecific binding. good luck

do you fix your particules on the grid before the primary antibody? I would try a 3% PFA (in PBS) fix 10 min, then 100mM glycine (5min to neutralize PFA), wash 2x 5 min PBS then incubate with primary and secondary antibodies as you did.

 I would ask is your grid coated and how; at least a Formvar coating is needed, better with a further carbon overlay.

I would advice you to try first to visualise your phages without the ICC. To me it seems your phages are absent from your pictures. Might have been removed by blotting, Try to apply small drops of phage suspensions (different dilutions) on the grids and just air-dry the drops before the staining.

In my experience, 2% UA is quite high and would make the biomaterial black; I use 0.5% for E. coli gold labeling because you need some transparancy to see the gold grains on the bug surface.

@ Delannoy, which concentration (mM) cacodylate-filtered buffer is used in 1% Glutaraldehyde (GA)?

@Asghari, negative staining before step 2 is working but phage structure still not so clear. 

@Bonhivers, next time I will try 3% PFA.

Usually 100 mM cacodylate, although if you are immunostaining I would probably fix in buffered paraformaldehyde (2-4 %) only.

Hi Shakhinur,

I agree with madam Stoitsova. You need to see the phage count (cfu) that best coats and confirm with negative staining. Carbon coated grids are best for this purpose.

If you fix with PFA you will need Ag retrieval. 

Blocking has to be min 30min.

Use fish skin gelatin instead of BSA which is particulate and doesnt filter..

Use 0.8% NRS in all your dilutions.

washing should be longer, maybe a minute.

Try staining with only 2% Lead citrate, may give better images..put the grid face down on the 50ul droplet.

Best,

Kaushiki

on second thoughts, Shakhinur, you could use 1% OsO4 as a fixative instead of PFA n then finally stain with Lead citrate at the end..