To keep our antibodies fresh and well-documented, is important that everybody follows the following rules:#
ALWAYS UPDATE THE ANTIBODY INVENTORY LOG when you order/receive antibodies or move an aliquot between different storage locations. The log is kept as a Google Document called ImmunoLog.
When you receive an antibody, please aliquot it immediately. Do NOT freeze unaliquotted stock tubes unless the storage instructions explicitly say it should not be aliquotted. Label aliquot tubes INDIVIDUALLY, preferably with the small round freezer dot stickers on the tops of the vials. Under no circumstances should an unlabeled tube be placed in any of the freezers. When you’re done, place the aliquots in one of our storage boxes with individual tube slots and update that information in the ImmunoLog. Do NOT store aliquots inside of a Falcon tube (which wouldn’t fit in the appropriate boxes anyway). Don’t add a new box to the freezer unless all existing boxes are completely full—there is probably plenty of space for your aliquots in an existing box. Note that you can use the ImmunoLog to count the total number of aliquots in a particular box without opening the freezer.
Unless the instructions say not to freeze them, all antibody aliquots should be stored in the -80° freezer (this includes antibodies which say “-20° C” or something similar for their storage instructions). The datasheets for all Alexa-conjugated secondary antibodies say they should be stored at +4°, but we keep them at -80° as well and they seem to work just fine.
When you need an antibody and there is none in the 4°C box, thaw an aliquot from storage at -80C and UPDATE THE IMMUNOLOG. Ideally, you should update the log just before you walk to the freezer, when you are looking at it to see which freezer box the aliquots you need are in. There should only be one tube of a given antibody at a time at 4°C. If for some reason you don’t like the tube that’s currently at 4°C, throw it away before thawing a new tube. If it’s not good enough for you, it shouldn’t be good enough for anyone else.
Keep the antibodies cold when you’re working with them. Either bring your tube up to the fridge and take out what you need quickly, or keep the source vial on ice at your bench. This will help us to remember to always return the antibodies to the fridge.
REMEMBER TO UPDATE THE IMMUNOLOG.
For any common antibody shared by the lab, when you get close to the last aliquot, re-order it. You should NEVER input “0” on the log and then walk away. There are basic items (i.e., common fluorophores for at least anti-mouse and anti-rabbit secondaries, streptavidin conjugates, etc.) that we should always have in the lab.
General procedures for making working stocks of antibodies:
To reconstitute Alexa Fluor-streptavidin conjugates, follow the manufacturer’s directions adding freshly filtered 1x PBS to make a solution of 2mg/mL, then add sodium azide to 0.05% final concentration (to inhibit microbial growth–the stock has the potential to go bad w/o; 5% solution lives in hood). Fluorescent avidin conjugates are supplied lyophilized in unit sizes of 5 mg, except the Alexa Fluor 488 conjugate, which is supplied in a unit size of 1 mg.
To store Alexa Fluor-conjugated secondary antibodies and streptavidin, mix, spin, and aliquot into ~100 microliter sub-stocks (or as much as we will go through in 3 months or less). One aliquot goes to the 4° box, the rest go to the -20° box. Get a new aliquot from the freezer if the one in the fridge is over 4 months old.
To make a working stock, mix and spin the stock then remove the needed amount. Before adding your working stock to your tissue samples do a hard spin (max rpm on tabletop microcentrifuge) for 5 minutes, in order to pellet any aggregated protein (so as to avoid artifactual speckling). Avoid pulling antibody solution from the bottom of the spun tube.