Getting Started
Growing Cells:
I prefer to grow my cells on 18mm diameter circular coverlips (fisherbrand 18CIR‐1, cat# 12‐545‐100) placed one per well of a 12 well culture dish plate. I calculate the cells needed and treat the 12 well dish like a T‐75 and split the 12 ml of media into 1ml/well.
You'll definitely need to sterilize you're coverslips under the cell culture hood. You can immerse them in 70% EtOH briefly, and drop them into the 12 well plates. Leave the wells open with the lid inside up and turn the germicidal UV that is standard in most cell culture hoods on for 15 minutes. This should be enough time to sterilize the plastic and glass and have the EtOH evaporate.
Handling Cells:
You will need to purchase a set of fine tipped forceps (the kind commonly used for handling electron microscopy grids, I would recommend getting at least 2 pair, invariably, one pair will get ruined because someone will use them for other purposes and bend the delicate tips!)
You can order them from Ted Pella at http://www.tedpella.com
Purchase the #7 biology grade, curved are easier for handling coveslips in the 12 wells. The brand is unimportant, just buy the cheapest #7's.
Incubating Cover Slips in Humidity Chambers:
You will construct a humidity chamber using some plasticware (I use a large 225mm square culture plate myself, but anything that you can cover and open easily without jarring the contents inside will work).
Place paper towels or filter paper on the bottom and wet until the paper is very moist but not sloshing water. Take parafilm and place over the wet paper. You want it wet enough, but not too wet that water may ride over the parafilm when moving the chamber from your desk to a dark place for incubation.
Your cells will be placed CELL SIDE DOWN on a 20‐80ul spot placed on the parafilm. Feel free to mark the parafilm w/ identifying characters regarding antibody or protocol as long as the writing NEVER comes in contact w/ the antibody solution. If you use 20 uls you WILL HAVE TO FLOAT the coverslips up with PBS or you will run the risk of ripping your cells off the coverslip when you pick them up. Do this by slowly adding PBS (about 40‐80uls or so) with a 200 ul pipettman to the side of the coverslip. Capillary action should wick PBS under the coverslip under, and you can now easily lift the coverglass off.
I usually change the parafilm between 1* and 2* antibody incubations just to be on the safe side unless there is plenty of room and you are processing just a few coverslips.
Wash the cells by placing them back in the corresponding 12 well CELL SIDE UP and using a 3‐4 ml volume for the washes.
Mounting your Cells and Mounting Media:
You will need to make mounting media, a 50:50 mix of PBS w/ Glycerol and 1% DABCO (Sigma) to retard photobleaching.
In a 15ml Conical cell culture tube, add 100 mg of DABCO to 5 ml of PBS and dissolve.
Add 5 ml of Glycerol (Glycerin) and gently invert until the two mix thoroughly.
This will last for a long time since you use about 4‐5 uls per 18mm coverlslip.
I usually clean a slide by exhaling on it and wiping of the condensate w/ a Kimwipe.
Place 4‐5 ul's of Mouting media on the center or a little of center (if you want to mount 2 coverslips per slide). Take your coverslip from the 12 well and line it vertically and touch the bottom to a paper towel to wick off any excess PBS. Now carefully place the coverslip CELL SIDE UP on the PAPER TOWEL to wick of any PBS on the back of the coverslip (you can press the center of the coverlip down gently with your forces in the closed position to make sure the back gets as dry as possible).
Now , carefully and gently place the coverslip on the spot of mounting media CELL SIDE DOWN. Using clear NAIL POLISH spot 4 "corners" of the circular coverslip with a drop (not too large) of nail polish 12,3,6,9 O'clock positions and allow to dry for a minute or so. Now carefully seal the edges with nail polish (if you try to seal the edges all at once without spotting the corners, you will find the coverslip will move around a bit and could grind your cells over the glass). There is a balance here, you want to get enough nail polish over the edge to allow a good seal, but not too much that you start to greatly diminish your viewing area by covering the coverslip with nail polish (the objective cannot view a field that is covered in nail polish).
Blocking Buffers and Antibody Incubations:
I usually use 2% defatted BSA in PBS for all my protocols (I use defatted BSA because I use that to make a complex with NBD‐Ceramide to stain the Golgi Complex, but any BSA will do). The Teleost Gelatin is an exceptional blocking buffer but you have to be careful with it, as it can block your specific sites as well, especially if you use it at higher concentrations (like 0.2% or above!)
Spinning down the antibodies is essential. I've noticed a big difference when I don't and my aliquot of 2*
is a little old.
As a supplier, I buy ALL of my fluorescent secondaries from Jackson Immunochemical http://www.jacksonimmuno.com/home/catalog/aff‐list.htm.
They are relatively inexpensive and have a wide range. You should buy the type that has NOT been minimized against any host species (they'll say: min X hum, goat etc.)
