Passive Cutaneous Anaphylaxis (PCA) (PDF)
Day 1
Passive Sensitization with anti-DNP IgE:
Inject 20ng IgE anti-DNP i.d. (in 20µl) and 20µl vehicle (HMEM-Pipes) in left/right ear (1cc syringe; 30G needle).
Day 2
Challenge with DNP-HSA:
Tail: Inject 100µg DNP-HSA i.v. (in 200µl in NaCl) (1cc syringe; 27G needle).
Eye: Inject 100µg DNP-HSA i.v. (in 100µl in NaCl) (1cc insulin syringe; 28G).
If using Evan’s blue, inject 100µg DNP-HSA in 1% Evan’s blue (in NaCl) i.v.
Readout:
Ear swelling: 0 (baseline), 0.5, 1, 2, 4 & 6h after challenge.
Histology: Sacrifice mice after 6h and take ears for histology (alkaline Giemsa of plastic (Eppon)-embedded 1µm sections and Giemsa or Toluidine Blue of paraffin-embedded sections).
Extravasation: Sacrifice mice after 30-60 min, take ears, cut them into 4-8 pieces and place them in Dimethylformamide (e.g. DriSolv from EMD). Incubate O/N at 37°C (or 3h at 55°C) and read OD @ 650nm.
HMEM-Pipes:
Sigma M4642 (MEM)
+ Pipes 0.47g
+ NaOH 0.105g
