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Protocol for Wholemount Immunohistochemistry of Adult Fly Tissue
Author: Jennifer Beck, updated date: , view: 12, Q&A: 0
Tags: Microscopy, Fly and Drosophila

Procedure

  1. Stock Solution:

    • PEM Solution:

      • 125 mM Pipes (pH 7.0)

      • 2.5 mM EGTA (pH 8.0)

      • 1.25 mM MgSO4

    • Fix Solution:

      • 4% paraformaldehyde (PFA) in PEM (freshely prepared)

        • PFA = 100 mg into 500 ul H2O + 1ul 1N NaOH, incubate ~70°C (heating block) for ~10min = 20%

        • 200ul PFA + 800 ul PEM = 4%PFA

    • TBS-TX:

      • 20 mM Tris-HCl (pH 7.5)

      • 130 mM NaCl

      • 1 mM EDTA

      • 0.1% Triton X-100 

    • TBS-TB: 

      • TBS-TX + 0.2% BSA 

  2. Dissect Flies in PBS (~300 ul) within 30min

  3. Fix with 250 ul of 4% PFA in PEM for 30 min

  4. Wash with 300 ul of PEM for 10 min x2 times

  5. Incubate with 300 ul of PEM containing 1% NP40 for 30 min

  6. Wash with 300 ul of TBS-TB for 5 min x2 times

  7. Blocking with 250 ul of TBS-TB containing 5% goat serum for overnight at 4°C or 2hrs at room temperature (RT)

  8. Incubate with 250 ul of primary antibody in TBS-TB for overnight at 4°C or 2hrs at RT

  9. Wash with 300 ul of TBS-TB for 10min 3x times

  10. Incubate with 250 ul of secondary antibody in TBS-TB for overnight at 4°C or 2hrs at RT

  11. Wash with 300 ul of TBS-TX for 10 min x3 times

  12. Rinse with 300 ul of PBS for 5 min x2 times

  13. mount on the slide glass with mounting medium containing DAPI


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