Procedures & recipes

Our public tricks are on the Tools page: Tools and Tricks

General:

    • Agarose is made in deionized water. More on making agarose here. Currently we are using 3.5% agarose (but in the past 3% has worked too).

    • Distilled water (ddH20, or milli-Q) has an expiration of 6 months, when it is placed in a squeeze bottle next to an animal/surgery setup.

    • Ringer's solution has an expiration of 6 months, when it is placed in a squeeze bottle next to an animal/surgery setup.

    • Recipes for stock solutions

Making Ringer with Dextrose:

There is currently a weekly plan to make only the necessary amount of solution as there is a saline shortage.

To make 10 mL of 5% Ringer-Dextrose:

  • 1mL Dextrose + 9mL saline

  • Using a syringe, remove the needed amount of saline from the bag and put into a conical falcon tube.

  • Using a fresh needle, remove the needed amount of dextrose from the bottle, add to the conical tube with saline and mix.

  • Make sure to label and date the fresh made ringer solution, including expiration date.

2-photon:

Recovering stainless steel material from dental cement:

    • For small material (such as mouse head posts), one can put the block of cement and the item in the acetone vial in the fume hood in room 327. Leave it there overnight, and use forceps to fish the piece out. Wash the piece and the forceps thoroughly with water. Check the piece to make sure it still works (for a head post, does it still fit in the chuck?) before returning the piece to its home in the lab.

Histology:

How to do stuff:

  • Testing the gain of the Multichannel Systems Amplifier

Preserved brains in jars:

    • Write what it is and what it is stored in "squirrel brain in 4% PFA" (abbreviating paraformaldehyde is okay)

    • Write the date of the experiment and the name of the experimenter

    • Indicate what hemisphere(s) were used in the experiment, and write down any relevant parameters (for example: "2 electrode penetrations with lesions in right hemisphere areas 17/18")

    • Store it in the cold room at the end of the hallway (Bassine 348)

Processed slides:

    • Each slide should be labeled with the animal information (such as experiment date), hemisphere, slide orientation (S/C/H for saggital, coronal, horizontal, use "para" if it applies), thickness, and slide number

    • Non-fluorescent slides can be stored at room temperature in drawer F3 in the histology room'

    • Fluorescent slides should be stored in the refrigerator

Using a F/Air canister:

  • What it is: The purpose of the F/AIR canister is to remove isofluorane from the output side of a gas-based anesthesia system when no building vacuum is available. This is the system we use in with mask or respirator-based anesthesia in Foster 123SX. In Bassine 329 and 322, we have connected the output of our gas-based anesthesia systems to the fume hood ventilation system, so we do not use F/AIR canisters in those rooms.

  • Proper use: The exhaust from the mask/respirator is passed through the F/AIR canister, and chemicals in the F/AIR canister trap the isofluorane within the canister. Before a new canister is used, it needs to be weighed (accurate to about 1 gram). As the canister accumulates isoflurane, its weight increases, and the canister needs to be replaced when the weight increases by some amount specified on the canister (usually 50 grams) so that it is effective at removing isoflurane.

  • NOTICE: It is _important_ that this is done. If we need more canisters, we can order them.

  • In the squirrel days, we weighed our F/AIR canisters and replaced them at the right time...I think for our usage this was usually monthly, and occasionally twice a month during heavy usage (which was 70 experiment-hours per week). But we went by the weight of the canister rather than time. We didn't weigh after every experiment, but we became familiar with the interval and weighed every experiment when it started getting close.

How to fill an isoflurane vaporizer (new model):

    • Find an isoflurane bottle. New bottles are kept in the locked drug cabinet, in-use bottles are kept in the fume hood. Open the bottle cap (use face mask to cover your nose).

    • You will find a long tube like attachment sitting on the iso vaporizer. At one end it has two different-sized grooves on a purple circular cap. Matching those grooves to the two different-sized ridges on the purple ring at the iso bottle mouth, attach the tube on to the iso bottle mouth and turn clockwise to tighten.

    • On the iso vaporizer, there is a solid metal bar inserted into a horizontal slot, you can unwind the screw on top and the bar will come loose and hang from a string. Make sure the bottom screw is not loosened.

    • now insert the purple rectangular shaped head of the tube attached to iso bottle into the slot so that the 2 holes on the tube head are facing down. Once pushed all the way in tighten the top screw again, lightly. Now the bottle is attached to the vaporizer through the tube.

    • Holding the bottle horizontal tilt it so that the liquid flows in to the vaporizer through the tube. Air bubbles will come into the bottle as long as fluid is going in.

    • Watch the level of iso inside the vaporizer through the window. Fill iso only up to the upper mark indicated on the window.

    • Once filled to level, unscrew from top and pull out the tube. Put the metal bar back in slot and tighten.

    • Take off the tube from bottle mouth and recap bottle. We don't wash the tube, just keep it back on the vaporizer. Check to make sure iso is not leaking from the vaporizer.

    • If some iso remains in the bottle, put it back in the fume hood.

How to pull pipettes for virus injection:

    • We use a vertical puller to pull long-tapered pipettes with tip diameter in the 25-35uM range. Up to 40uM is usable, but pipettes with wider tips and shorter taper cause more damage to the brain.

    • Currently the lab's vertical puller is broken. We use a Narishige puller in the Goldstein lab. They have two pullers, an older Black one, and right next to it a newer model that is Green in color. Use the green one.

    • Use the complete set of weights attached, do NOT take off any weights.

    • Take a Drummond capillary and insert it into the groove of the puller from top, position it such that almost equal lengths of the capillary are left on either side of the filament. Close the protective door in front.

    • Do NOT press Red "start" button.

    • There is a black knob with 4 settings on it: step 1, step 2, No. 1 heater, No. 2 heater. Turn the knob to "No. 1 heater" position, then use the black dial for Heater 1 to set the heat around 63 (may need to vary between 61-64). It should start immediately heating up the filament.

    • Once it finishes pulling, turn the black knob back to "step 2" setting to turn it off.

    • Carefully remove your 2 pipettes. usually both the top and the bottom are usable.

    • Wait about a minute before pulling the next one, the filament tends to overheat sometimes and needs a bit of time to cool down.

    • These pipettes should have a nice long taper, but the tips will be closed, you will have to break the tip open. One way to do so is to clip it carefully with a pair of microscissors very near the tip to get the desired tip diameter. Sometimes just touching it carefully on a kimwipe could do the trick too.

    • Normally after we break the tip we check it under the super-old Zeiss scope in the perfusion room. Use 40x lens.

    • If the tip is seen to be within the 25-35uM range under the scope, we then bevel it using the Narishige Beveler in the tgf facility.