VTA Biocytin protocol adapted from Swietek et al - KravitzLab/KreedLabWiki GitHub Wiki

VTA Biocytin protocol adapted from Swietek et al.docx

Biocytin protocol adapted from Swietek et al., JOVE 2016.

Patching.

  • Use a potassium gluconate internal, if possible (“M”). The ‘thinner’ the slices are the better, so a max of 220 µM. 2mg of biocytin powder should be added to 1mL of internal solution to make a 0.2% biocytin solution. [NOTE: we now add 3-5 mg of biocytin for better reconstruction]
  • Patch and do your experiment as you would; whole cell configuration should be maintained for at least 10 minutes.
  • When you’re done, clamp the cell at positive (+40mV) while slowing moving your pipette away from the cell in small steps up and back, up and back. Try not to apply positive pressure unless you really need to (if your cell is being dragged through the tissue). Excessive positive pressure will cause biocytin to spread in the slice, making it hard to identify the filled cell.
  • Leave the slice left in the bath for an additional 3-5 minutes to allow solution to diffuse throughout processes (if you’re interested in that morphology especially)
  • Transfer the slices to a 24-well plate containing 4% PFA for fixation, do not put rig tools in the PFA.
  • Between 24 - 48 h after PFA fixation, transfer the slices to 0.1 M Phosphate-buffered Saline (PBS) for storage prior to immunostaining. Ideally, biocytin recovery and immunostaining are best when performed soon after fixation, although staining performed within 7 d of fixation yields good results.

Staining the primary antibodies

  • Wash the tissue 3x 10 min in PBS

  • Block with 10% blocking serum (normal goat or donkey) diluted in 0.1% Triton-X-100 in PBS for 2h at room temperature. `

  • Incubate sections in primary antibodies with 3% blocking serum and 0.1% triton-X-100 in PBS for ~48 hours at 4°C. N.B. You can re-use this soup 2-3 times without issue.

Note: You can recover the antibody soup from the incubation wells, transfer to an Eppendorf tube and store at 4°C for future use (1-3 more times).

Staining the secondary antibodies and biocytin

  • Wash the brain sections 3 x 15 min in 0.1 M PBS.
  • Incubate the sections in streptavidin blue dy conjugate (1:1000, emission in the UV spectrum), 3% blocking serum and 0.1% triton-X-100 in PBS in 4C overnight. * The secondary antibodies of interest should also be added here.

Note: You can re-use this soup 2-3 times without issue:

CAUTION: PFA is carcinogenic, and appropriate personal protective equipment, including gloves and a face mask, must be used to avoid skin irritation and inhalation. PFA is flammable and must be kept out of reach of fire. PFA is never disposed of in the drain and must be collected as chemical waste. PFA fixation must be isolated from areas utilized for live slice preparation to avoid contamination of the physiology setup, including transfer pipettes.

NOTE: Slices can be maintained in PBS with 0.02 - 0.05% sodium azide for staining performed up to and over 90 days after fixation. Although overnight fixation in PFA works well for most antibodies, it is possible that some antibodies work best when the duration of incubation in fixative is reduced. Fixation in PFA for over 48 h reduces the availability of antigens and diminishes the chances of successful secondary immunostaining for neurochemical markers.

CAUTION: Sodium azide is extremely hazardous and an irritant upon contact with the skin or eyes or upon ingestion or inhalation. Severe over-exposure can result in death. The carcinogenicity and mutagenicity of the compound are not known. Use appropriate personal protective equipment including gloves, splash goggles, a lab coat, and a dust respirator. Sodium azide is never disposed of in the drain and must be collected as chemical waste

Primary and secondary antibody