Langerhans Cell Staining

DC labeling of epidermal sheets.
[Modified from Aluwihare et al., 2009 Jan 15;122(Pt 2):227-32]
 
  1. Shave mouse back with electric razor.
  2. Apply NEET. Wait 5-10 min.
  3. Wet paper towel and wipe off cream.
  4. Kill (CO2).
  5. Prepare P80 with PBS.
  6. Remove skin and put in PBS (same for ears).
  7. Let sit in PBS for a couple of minutes and wash (1-2 changes of fresh PBS to get rid of NEET).
  8. With the dermal side up, scrape with spatula (clean well). Wet occasionally with PBS (e.g. dip spatula in PBS). When done, float in PBS (epidermis up).
  9. Ears: split open between two layers. This is tricky. One way is to insert scissors into ear at cut site, then cut up one side. After opening up a side a bit like this, then use 2 forceps to grip the two sides of the ear and pull apart. The ear will gradually open up. When done, float in PBS (epidermis up).
  10. Transfer into dish with 2M NaBr. Epidermal side up (scraped side down), spread up. Incubate @37oC for 30-45 min (ears usually take less time). Try after 30 min, if not ready, incubate longer.
  11. Put PBS in dishes. Transfer from NaBr into PBS.
  12. Spread on surface, epidermal side up (wet surface). Wet the sample with some drops of PBS.
  13. Make punch. Remove with tweezers, float on PBS. This is difficult. Most successful way seems to be to use a fine forceps to gradually push the epidermis back from one side, pushing the whole sheet toward the opposite side of the punch circle, then grasp the piece of epidermis and transfer to PBS. Sometimes the sheet is initially folded over itself, and can be rescued carefully poking at it, or lifting/spreading it from below, or even pulling on it with two forceps from opposite sides, until it opens up flat. Try to get 3-5 sheets. Keep floating samples on ice.
  14. Label ceramic plates.
  15. Put samples floating in 95% EtOH for 20-30 min.
  16. Transfer to PBS (wash briefly in PBS). after fixation can store in PBS at 4oC for several days.
  17. Incubate with primary antibody 60-90 min @RT. Carefully remove PBS, then add 200-400 l of the primary Ab in 0.5 M NaCl/PBS. 1:100 dilution, biotin-conjugated rat anti-mouse-A/I-E monoclonal Ab, #06342 Pharmingen.
  18. Wash 3X in PBS.
  19. Incubate with HRP-avidin 1h@RT. HRP-avidin is ready to se. NOTE: alternatively, use the ABC kit.
  20. Wash 3-4X in PBS.
  21. Incubate with DAB solution (8 min). Wash in PS, then water.
  22. Mount.