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Neuron immunohistochemisry

Fatih Kocabaş

Moleküler Biyolog ve Genetikçi

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miRNA cloning
Chlamy protocol
Neuron Labelling

Protocol for Labeling of Neurons cells - immunohistochemistry

  1. Take slides out from -20
  2. Let them dry at RT, not more then 10 min
  3. Use Pap pen (contains oily substance) to place a barrier around the sections
  4. Rehydrate slides with 1x PBS for 5 min
  5. Using suction dry around sections, but not sections directly
  6. Then wash with 1x PBS+ 0.2 NP-40 (or Triton) solution for 5 min, 3 times
  7. Then block with 1% Normal Donkey Serum (50 ul serum + 5 ml 1x PBS+ 0.2 NP-40) at least 30 min (40 min-1 hr)
  8. Apply primary antibodies ON (approx. 16 hr) at 4C, 200 ul is enough for 9 sections
    NEUN is mouse antibody, general marker for neurons: You can use 594 or 568 nm secondary antimouse, but 594 is preferred; dilution is 1:500 in PBS for secondary antibody
    for YPF, we have rabbit antibody: For the secondary antibody use 488nm antirabbit , dilution is 1:500 in PBS for secondary antibody
  9. Wash slides with 1x PBS for 5 min, 3X
  10. Apply fluorescently labelled secondary antibodies, 1:500 dilution, 200 ul for one slide, and keep in humidity chamber for at least 1 hr up to 2 hrs.
  11. Remove sec ab
  12. Wash with 1x PBS for 5 min, 3X
  13. Remove PBS and apply 1-2 drops of VectaShield solution
  14. Place appropriately sized coverslip
  15. Dry excess solution by putting slides facing down onto tissue paper
  16. Seal with nail polish if you want to store them for an extended time
  17. Store in slide holder at 4C until microscopy