ACN

I have had excellent results with Ribozol.  Cheap and efficient.

This: http://www.piercenet...e-second-Ab.pdf will give you a good overview.  We usually get our labelled secondaries from Jackson Immunoresearch...they are reliable and the prices are quite reasonable.

I also work with MCMV.
We fix and stain our plaque assays using methylene blue with 10% formaldehyde. I've posted your protocol below, and put in where mine differs:

Set up NIH 3T3 cells in 6 well plates (500k cells/well) [i]I do mine in 24 well trays, less cells to use, more dilutions per plate etc[/i]
Next day, whe cells should be 90% confluent I wait for my cells to be 100% confluent
Thaw and sonicate virus for 6 seconds twice. Store on ice don't know why you are sonicating here?
Make a series of 10-fold virus dilutions --- 10-1 to 10-7
Aspirate media from each well and add 2ml of diluted virus to each well. I use 200ul for a 24 well
Add 2mls of media (no virus) to one well for negative control.
Spin the plate at 100g for 20 min, rotate the plate and spin again at 100g for 20 min I don't do this, are you trying to centrifugally enhance here? Why are you centrifuging? I incubate for 1 hour at 37C
Aspirate the innoculum and add 3ml of overlay per well

Overlay: I use methylcellulose rather than agarose

100ml
45ml 2X DMEM (Final 1X)
45mls of 1% LMP agarose (0.75%)
10ml FCS (10%)  This is too high, you don't want your cells to be growing once inoculated with virus, you should have a final concentration of 2% instead
4.5ml of 7.5% Sodium Bicarbonate (0.33%)

Plate a duplicate set of plates, but use an overlay with 2% FBS why?

after incubation for 3-5 days, we add about 1ml of the methylene blue with 10% formaldehyde, leave at RT overnight then wash off (with the overlay) the next day

If you are just wanting to visualise the plaques, you can see them by illuminating the plate from the side and holding the plate over a dark background.  Otherwise, you can add neutral red to the overlay, you will get clear spots developing where the dye is excluded by the dying cells.

THE ROCK, on Jul 10 2009, 06:59 AM, said:

HMG, on Sep 9 2009, 09:46 AM, said:

Buddy, on Dec 6 2009, 04:53 PM, said:

If you all read the above posts you will see that you are in error... re-using is a problem, if you do it you are jeopardizing you results through a number of factors including cellular damage, cell type drift (lab evolution), and general contamination issues.

Be better scientists!